Methods and compositions using listeria for adjuvant treatment of cancer

ABSTRACT

Provided herein are prime-boost regimens and materials used therein. The prime-boost regimens enhance the immune response to a target antigen. The vaccines used for boost are comprised of recombinant attenuated metabolically active  Listeria  that encodes an expressible antigen that is cross-reactive with the target antigen. In some examples, the immune response is a cellular immune response.

This application is filed under 35 U.S.C. §371 as the U.S. national phase of International Application No. PCT/US2011/037602, filed May 23, 2011, which designated the U.S. and claims the benefit of priority to U.S. provisional patent application 61/347,447 filed May 23, 2010, which is hereby incorporated in its entirety including all tables, figures and claims.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of National Cancer Institute Grant No. NHI 1 K23CA104160-01.

BACKGROUND OF THE INVENTION

It is thought to be desirable to establish strong cellular immunity to specific pathogens. Repeated administrations with the same vaccine (homologous boosting) have proven effective for boosting humoral responses. However, this approach is relatively inefficient at boosting cellular immunity because prior immunity to the vector tends to impair robust antigen presentation and the generation of appropriate inflammatory signals. One approach to circumvent this problem has been the sequential administration of vaccines that use different antigen-delivery systems (heterologous boosting). This strategy is referred to as “prime-boosting”.

The following are examples of heterologous prime-boost regimens. Those involving a DNA prime include: a DNA prime: DNA prime/bacterial boost (Listeria) against viral antigens (Boyer, et al. (2005) Virology 333:88-101); a DNA prime/bacterial vector (Bacillus) boost, against bacterial antigen (Ferraz, et al. (2004) Infection Immunity 72:6945-6950); a DNA prime/viral vector boost, against tumor antigens (Goldberg, et al. (2005) Clin. Cancer Res. 11:8114-8121; Smith, et al. (2005) Int. J. Cancer 113:259-266); a DNA prime/viral boost against viral antigens (Toussaint, et al. (2005) Vaccine 23:5073-5081; Cebere, et al. (2006) Vaccine 24:417-425; Coupar, et al. (2006) Vaccine 24:1378-1388); a DNA prime/protein boost against viral antigens (Cristillo, et al. (2006) Virology 346:151-168; Rasmussen, et al. (2006) Vaccine 24:2324-2332); a DNA prime/viral boost, against antigens of a parasite (Gilbert, et al. (2006) Vaccine 24:4554-4561; Webster, et al. (2005) Proc. Natl. Acad. Sci. USA 102:4836-4841); DNA prime/adjuvanted protein boost, against tumor antigens (Prud'homme (2005) J. Gene Med. 7:3-17); DNA prime/viral boost plus protein boost, against viral antigens (Stambas, et al. (2005) Vaccine 23:2454-2464); and DNA prime (nanoparticles)/protein boost, against viral antigen (Castaldello, et al. (2006) Vaccine 24:5655-5669).

The following heterologous prime-boost regimens utilize prime compositions not involving DNA: Dendritic cell (DC) prime/bacterial (Listeria) boost, and DC prime/viral boost, against bacterial antigens (Badovinac, et al. (2005) Nat. Med. 11:748-756); bacterial vector prime (Salmonella)/protein boost, against bacterial antigens (Vindurampulle, et al. (2004) Vaccine 22:3744-3750; Lasaro, et al. (2005) Vaccine 23:2430-2438); adjuvanted protein prime/DNA boost, against viral antigens (Sugauchi, et al. (2006) J. Infect. Dis. 193:563-572; Pal, et al. (2006) Virology 348:341-353); protein prime/bacterial vector (Salmonella) boost, against viral antigens (Liu, et al. (2006) Vaccine 24:5852-5861); protein prime/viral vector boost, against viral antigen (Peacock, et al. (2004) J. Virol. 78:13163-13172); Heterologous viral prime/viral boost, using different viral vectors, against viral antigens or tumor antigens (Ranasinghe, et al. (2006) Vaccine 24:5881-5895; Kaufman, et al. (2004) J. Clin. Oncol. 22:2122-2132; Grosenbach, et al. (2001) Cancer Res. 61:4497-4505) Heterologous prime/boost using lipid vesicles, against bacterial antigens (Luijkx, et al. (2006) Vaccine 24:1569-1577).

A reagent that is useful for modulating the immune system is Listeria and particularly Listeria monocytogenes (L. monocytogenes). L. monocytogenes has a natural tropism for the liver and spleen and, to some extent, other tissues such as the small intestines (see, e.g., Dussurget, et al. (2004) Ann. Rev. Microbiol. 58:587-610; Gouin, et al. (2005) Cum Opin. Microbiol. 8:35-45; Cossart (2002) Int. J. Med. Microbiol. 291:401-409; Vazquez-Boland, et al. (2001) Clin. Microbiol. Rev. 14:584-640; Schluter, et al. (1999) Immunobiol. 201:188-195). Where the bacterium resides in the intestines, passage to the bloodstream is mediated by listerial proteins, such as ActA and internalin A (see, e.g., Manohar, et al. (2001) Infection Immunity 69:3542-3549; Lecuit, et al. (2004) Proc. Natl. Acad. Sci. USA 101:6152-6157; Lecuit and Cossart (2002) Trends Mol. Med. 8:537-542). Once the bacterium enters a host cell, the life cycle of L. monocytogenes involves escape from the phagolysosome to the cytosol. This life cycle contrasts with that of Mycobacterium, which remains inside the phagolysosome (see, e.g., Clemens, et al. (2002) Infection Immunity 70:5800-5807; Schluter, et al. (1998) Infect. Immunity 66:5930-5938; Gutierrez, et al. (2004) Cell 119:753-766); L. monocytogenes’ escape from the phagolysosome is mediated by listerial proteins, such as listeriolysin (LLO), PI-PLC, and PC-PLC (see Portnoy, et al. (2002) J. Cell Biol. 158:409-414).

In contrast to the immunotherapy approaches discussed above, in which immunotherapy is used as a primary treatment, adjuvant therapy refers to the use of a secondary treatment prior to (“neoadjuvant”) or following (“adjuvant”) a primary therapy such as surgery or radiation, where the primary therapy is intended to remove or destroy the primary tumor. By way of example, adjuvant therapy may be given after surgery where the detectable disease has been removed, but where there remains a statistical risk of relapse due to occult disease. Adjuvant systemic therapy and radiotherapy are often given following surgery for many types of cancer, including colon cancer, lung cancer, pancreatic cancer, breast cancer, prostate cancer, and some gynaecological cancers.

SUMMARY OF THE INVENTION

The invention provides adjuvant therapy strategies employing Listeria bacteria for secondary treatment of cancer. Before or after the subject has been administered one or more primary treatments, an effective dose of a Listeria vaccine expressing a cancer-related antigen is administered. The invention includes kits containing the Listeria-based vaccine packaged in suitable containers, and may include instructions.

In a first aspect, the present invention relates to methods for adjuvant treatment of a mammal suffering from cancer, the method comprising administering to the mammal an effective amount of a composition comprising an attenuated, metabolically active Listeria that encodes an expressible, immunologically active portion of a cancer antigen, where the administration is provided as an adjuvant treatment prior to or following a primary therapy administered to the mammal to remove or kill cancer cells expressing the cancer antigen.

As noted above, the compositions of the present invention may be provided as a neoadjuvant therapy; however in preferred embodiments, the compositions of the present invention are administered following the primary therapy. In various embodiments, the primary therapy comprises surgery to remove the cancer cells from the mammal, radiation therapy to kill the cancer cells in the mammal, or both surgery and radiation therapy.

As Listeria can be a pathogenic organism, and particularly in the immunocompromised, it is preferred that the administration step comprises administering the attenuated, metabolically active Listeria that encodes an expressible, immunologically active portion of a cancer antigen in multiple doses. “Attenuation” refers to a process by which a bacterium is modified to lessen or eliminate its pathogenicity, but retains its ability to act as a prophylactic or therapeutic for the disease of interest. Bacterial attenuation can be achieved by different mechanisms. One is to introduce mutations into one or more metabolic pathways, the function of which is essential for bacteria to survive and grow in vivo to cause disease. In the case of Listeria, in certain embodiments the bacterium is mutated to lessen or prevent the ability to grow and spread intracellularly. Preferred Listeria can comprise a mutation that inactivates ActA; a mutation that inactivates InlB; or both. Most preferably, an attenuated, metabolically active Listeria which is deleted for both its native ActA and inlB genes (ΔactAΔinlB) is employed in the present invention. In certain embodiments, the methods employ a Listeria that is killed but metabolically active (“KBMA”).

One or more nucleic acids encoding any of a variety of cancer antigens are provided for recombinant expression by the compositions of the present invention. In certain embodiments, the cancer antigen is all or a portion of mesothelin. Other suitable antigens are described in detail hereinafter, and may depend on the type of cancer being treated and the antigens being expressed by that cancer. In various embodiments, the cancer being treated is selected from the group consisting of pancreatic cancer, non-small cell lung cancer, ovarian cancer, and mesothelioma. This list is not meant to be limiting.

In addition to the primary therapy, which is preferably surgery or radiation as described, the compositions of the present invention may be delivered as part of a prime-boost regimen. For example, prior to administration of the Listeria compositions of the present invention, the mammal may previously be administered cells from a human cell line expressing the cancer antigen of interest, wherein said cells have been recombinantly modified to produce and secrete granulocyte macrophage colony stimulating factor, and wherein said cells have been modified by radiation so as to prevent the cells from dividing (i.e., GVAX® vaccine, Cell Genesys, Inc.).

It is to be understood that the invention is not limited in its application to the details of construction and to the arrangements of the components set forth in the following description or illustrated in the drawings. The invention is capable of embodiments in addition to those described and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the abstract, are for the purpose of description and should not be regarded as limiting.

As such, those skilled in the art will appreciate that the conception upon which this disclosure is based may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing OVA specific cellular immune responses to a target antigen (OVA) resulting from prime-boost regimens using Listeria monocytogenes and Vaccinia virus expressing the antigen. OVA-specific responses in vaccinated C57BL/6 mice using OVA257-264 peptide (SIINFEKL (SEQ ID NO:1)) in ICS assays are shown.

FIG. 1B is a graph showing human Mesothelin-specific cellular immune responses resulting from prime-boost regimens using Listeria monocytogenes and Adenovirus expressing the target antigen (human Mesothelin).

FIG. 2 is a graph showing human Mesothelin-specific cellular immune responses in Balb/c mice resulting from prime-boost regimens using Listeria monocytogenes, Adenovirus and Vaccinia virus. Prime-boost regimens are also illustrated.

FIG. 3A is a graph showing the human Mesothelin-specific cellular immune responses in C57BL/6 (HLA-A2 transgenic) mice resulting from prime-boost regimens using increasing levels of Adenovirus prime and a constant level of Listeria monocytogenes boost. (Splenocytes were stimulated with mesothelin peptide pool.)

FIG. 3B the human Mesothelin-specific cellular immune responses in Balb/c mice resulting from prime-boost regimens using increasing levels of Adenovirus prime and a constant level of Listeria monocytogenes boost. (Splenocytes were stimulated with mesothelin peptide pool.) The prime-boost regimens are also illustrated.

FIG. 4A shows on the left a graph showing the human Mesothelin-specific cellular immune response resulting from an adenovirus prime in mice with a pre-existing immunity to adenovirus. Also shown in FIG. 4A (on the right) are the adenovirus-specific responses as measured using the class I Hex3 epitope (an adenovirus-specific epitope). The prime-boost regimens are also illustrated.

FIG. 4B is a graph showing the human Mesothelin specific cellular immune response and the titer of neutralizing adenovirus-specific antibodies resulting from an Adenovirus prime and Listeria monocytogenes boost in mice with a pre-existing immunity to Adenovirus. The prime-boost regimens are also illustrated.

FIG. 5 is a graph showing AH1-specific cellular immune response resulting from a prime with either GVAX® (CELL GENESYS, INC.) or Listeria monocytogenes encoding AH1 and a boost with either GVAX® (CELL GENESYS, INC.) or the Listeria monocytogenes encoding AH1.

FIG. 6A is a graph showing the percent of Mesothelin₁₃₁₋₁₃₉ specific response among CD8⁺ T cells resulting from a prime with dendritic cells pulsed with Mesothelin peptide 131-139 or Adenovirus encoding human Mesothelin and boosted with Listeria monocytogenes encoding human Mesothelin. Prime occurred on day 0, boost occurred on day 8, and splenocytes were harvested on day 13.

FIG. 6B is a graph showing the absolute number of Mesothelin131-139 specific CD8+ T cells per spleen resulting from a prime with dendritic cells pulsed with Mesothelin peptide 131-139 or Adenovirus encoding human Mesothelin and boosted with Listeria monocytogenes encoding human Mesothelin. Prime occurred on day 0, boost occurred on day 8, and splenocytes were harvested on day 13.

FIG. 6C is a graph showing the percent of Mesothelin₁₃₁₋₁₃₉ specific response among CD8⁺ T cells resulting from a prime with dendritic cells pulsed with Mesothelin peptide 131-139 and boosted with one of the following Mesothelin encoding agents: Listeria monocytogenes, Adenovirus, or Vaccinia virus.

FIG. 7A is a graph showing the mouse Mesothelin specific immune response in splenocytes where the immune response was assessed for each peptide in the mouse Mesothelin peptide library. Balb/c mice received a prime with a naked DNA vector encoding mouse mesothelin, and a boost with Listeria monocytogenes encoding mouse Mesothelin.

FIG. 7B is a graph showing the mouse Mesothelin specific immune response in splenocytes where the immune response was assessed for each peptide in the mouse Mesothelin peptide library. Balb/c mice received a prime with adenovirus encoding mouse Mesothelin and a boost with Listeria monocytogenes encoding mouse Mesothelin. The prime-boost regimens are also illustrated.

FIG. 7C is a half-tone reproduction of a photograph of wells showing the results of elispot assays where splenocytes obtained from the mice (the study results of which are shown in FIG. 7B) were exposed to Mesothelin peptide nos. 278 (SEQ ID NO:2), 279 (SEQ ID NO:3), or 280 (SEQ ID NO:4).

FIG. 8 is a graph showing the OVA specific cellular immune responses resulting from prime-boost regimens using KBMA Listeria monocytogenes encoding OVA as prime, and as boost KBMA Listeria monocytogenes, Vaccinia virus, or “live” Listeria monocytogenes. All of the “boost” vectors encoded OVA.

FIG. 9A shows AH- and AH1/A5-specific T cell responses in vaccinated mice.

FIG. 9B shows Mesothelin-specific T cell responses in vaccinated mice.

FIG. 9C shows survival data for vaccinated mice following CT26 tumor challenge.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to materials and to methods for eliciting an immune response to a target antigen that is not a Listerial antigen. The target antigen is preferably one associated with a disease, such that an immune response to the target antigen will provide a therapeutic effect. The present invention provides vaccine sets for adjuvant treatment of cancer, where the Listeria-based vaccine is metabolically active Listeria that encodes an immunologically active portion of a target antigen that is expressed in the host to which it is administered.

The present invention is based, in part, on the findings that a number of primary cancer treatments may be enhanced with adjuvant use of vaccines comprised of live attenuated Listeria monocytogenes encoding a cancer-related antigen such as mesothelin.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques including those of molecular biology (including recombinant techniques), immunology, cell biology, biochemistry, and pharmaceutical practice. Such techniques are explained fully in the literature, for example, Molecular Cloning: A Laboratory Manual, second edition, (Sambrook et al.); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds); Current Protocols in Immunology (John Wiley & Sons, Inc., N.Y.), Handbook of Pharmaceutical Excipients (Rowe et al., Eds); Vaccines (Plotkin and Orenstein, 2003); and Vaccine Protocols (Methods in Molecular Medicine)(Robinsin, Cranage and Hudson, 2003).

DEFINITIONS

Abbreviations used to indicate a mutation in a gene, or a mutation in a bacterium comprising the gene, are as follows. By way of example, the abbreviation “Listeria ΔactA” means that part, or all, of the actA gene was deleted. The delta symbol (A) means deletion. An abbreviation including a superscripted minus sign (Listeria actin means that the actA gene was mutated, e.g., by way of a deletion, point mutation, or frameshift mutation, but not limited to these types of mutations. Exponentials may be abbreviated, for example, “3e7” means 3×10⁷.

As used herein, including the appended claims, the singular forms of words such as “a,” “an,” and “the,” include their corresponding plural references unless the context clearly dictates otherwise. All references cited herein are incorporated by reference in their entirety to the same extent as if each individual publication, sequences accessed by a GenBank Accession No., patent application, patent, Sequence Listing, nucleotide or oligo- or polypeptide sequence in the Sequence Listing, as well as figures and drawings in said publications and patent documents, was specifically and individually indicated to be incorporated by reference.

“Administration,” as it applies to a human, mammal, mammalian subject, animal, veterinary subject, placebo subject, research subject, experimental subject, cell, tissue, organ, or biological fluid, refers without limitation to contact of an exogenous ligand, reagent, placebo, small molecule, pharmaceutical agent, therapeutic agent, diagnostic agent, or composition to the subject, cell, tissue, organ, or biological fluid, and the like. “Administration” can refer, e.g., to therapeutic, prophylactic, pharmacokinetic, research, placebo, and experimental methods. “Administration” to a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell. “Administration” also encompass in vitro and ex vivo methods, e.g., of a cell, by a reagent, binding composition, or by another cell. The outcome of an administration can be assessed by, for example, increased survival time (e.g., to a life threatening proliferative disorder), decrease in tumor size, decrease in tumor number, decrease in metastasis from a specific tissue, decrease in metastasis to a specific tissue, decrease in titer of an infective agent, and the like, as compared with a placebo administration or with no administration. “Treatment” encompasses an administration where there is an expected efficacy. “Treatment” includes preventative (prophylactic) and therapeutic administrations.

“Antigen presenting cells” (APCs) are cells of the immune system used for presenting antigen to T cells. APCs include dendritic cells, monocytes, macrophages, marginal zone Kupffer cells, microglia, Langerhans cells, T cells, and B cells (see, e.g., Rodriguez-Pinto and Moreno (2005) Eur. J. Immunol. 35:1097-1105). Dendritic cells occur in at least two lineages. The first lineage encompasses pre-DC1, myeloid DC1, and mature DC1. The second lineage encompasses CD34⁺⁺CD45RA⁻ early progenitor multipotent cells, CD34⁺⁺CD45RA⁺ cells, CD34⁺⁺CD45RA⁺⁺CD4⁺ IL-3Ralpha⁺⁺ pro-DC2 cells, CD4⁺CD11c⁻ plasmacytoid pre-DC2 cells, lymphoid human DC2 plasmacytoid-derived DC2s, and mature DC2s (see, e.g., Gilliet and Liu (2002) J. Exp. Med. 195:695-704; Bauer, et al. (2001) J. Immunol. 166:5000-5007; Arpinati, et al. (2000) Blood 95:2484-2490; Kadowaki, et al. (2001) J. Exp. Med. 194:863-869; Liu (2002) Human Immunology 63:1067-1071; McKenna, et al. (2005) J. Virol. 79:17-27; O'Neill, et al. (2004) Blood 104:2235-2246; Rossi and Young (2005) J. Immunol. 175:1373-1381; Banchereau and Palucka (2005) Nat. Rev. Immunol. 5:296-306).

“Attenuation” and “attenuated” encompasses a bacterium, virus, parasite, infectious organism, tumor cell, gene in the infectious organism, and the like, that is modified to reduce toxicity to a host. The host can be a human or animal host, or an organ, tissue, or cell. The bacterium, to give a non-limiting example, can be attenuated to reduce binding to a host cell, to reduce spread from one host cell to another host cell, to reduce extracellular growth, or to reduce intracellular growth in a host cell. Attenuation can be assessed by measuring, e.g., an indicator of toxicity, the LD₅₀, the rate of clearance from an organ, or the competitive index (see, e.g., Auerbuch, et al. (2001) Infect. Immunity 69:5953-5957). Generally, an attenuation results an increase in the LD₅₀ by at least 25%; more generally by at least 50%; most generally by at least 100% (2-fold); normally by at least 5-fold; more normally by at least 10-fold; most normally by at least 50-fold; often by at least 100-fold; more often by at least 500-fold; and most often by at least 1000-fold; usually by at least 5000-fold; more usually by at least 10,000-fold; and most usually by at least 50,000-fold; and most often by at least 100,000-fold.

“Attenuated gene” encompasses a gene that mediates toxicity, pathology, or virulence, to a host, growth within the host, or survival within the host, where the gene is mutated in a way that mitigates, reduces, or eliminates the toxicity, pathology, or virulence. “Mutated gene” encompasses deletions, point mutations, insertion mutations, and frameshift mutations in regulatory regions of the gene, coding regions of the gene, non-coding regions of the gene, or any combination thereof.

“Cancerous condition” and “cancerous disorder” encompass, without implying any limitation, a cancer, a tumor, metastasis, angiogenesis of a tumor, and pre-cancerous disorders such as dysplasias. A mammalian subject with a cancer, tumor, pre-cancerous condition, pre-cancerous disorder, or cancerous disorder, and the like, encompasses a mammalian subject that comprises the cancer, per-cancerous disorder, or tumor, but also encompasses a mammalian subject where the tumor has been removed, where the cancer has been apparently eliminated (e.g., by chemotherapy or surgery or solely by the subject's immune system).

“Effective amount” as used in treatment encompasses, without limitation, an amount that can ameliorate, reverse, mitigate, or prevent a symptom or sign of a medical condition or disorder. Unless dictated otherwise, explicitly or otherwise, an “effective amount” is not limited to a minimal amount sufficient to ameliorate a condition, or to an amount that results in an optimal or a maximal amelioration of the condition. “Effective amount” within the context of administration of a prime and/or boost is that which causes an immune response in the mammal.

An “extracellular fluid” encompasses, e.g., serum, plasma, blood, interstitial fluid, cerebrospinal fluid, secreted fluids, lymph, bile, sweat, fecal matter, and urine. An “extracellular fluid” can comprise a colloid or a suspension, e.g., whole blood.

“Growth” of a Listeria bacterium is a term of the listerial art that encompasses intracellular growth of the Listeria bacterium, that is, growth inside a host cell such as a mammalian cell. While intracellular growth (“growth”) of a Listeria bacterium can be measured by light microscopy, fluorescent microscopy, or colony forming unit (CFU) assays, growth is not to be limited by any technique of measurement. Biochemical parameters such as the quantity of a listerial antigen, listerial nucleic acid sequence, or lipid specific to the Listeria bacterium, can be used to assess growth. A gene that mediates growth is one that specifically mediates intracellular growth. A gene that specifically mediates intracellular growth encompasses, but is not limited to, a gene where inactivation of the gene reduces the rate of intracellular growth but does not detectably reduce the rate of extracellular growth (e.g., growth in broth), or a gene where inactivation of the gene reduces the rate of intracellular growth to a greater extent than it reduces the rate of extracellular growth. To provide non-limiting examples, a gene that mediates intracellular growth is one where its deletion cuts down intracellular growth to less than 50%, to less than 40%, to less than 30%, to less than 20%, or to less than 10%, the intracellular growth exhibited by a wild type Listeria. To provide further non-limiting examples, a gene that mediates intracellular growth encompasses one where its deletion cuts down intracellular growth to less than 50% the intracellular growth of the wild type Listeria, but only cuts down extracellular growth to about 95%, 90%, 85%, or 80% that found with wild type Listeria. In this context, the term “about” refers to plus or minus 5%.

“Killed but metabolically active” (KBMA) encompasses any bacterium that contains a modified genome, for example, where the genomic modification is sufficient to prevent colony formation, but where the genomic modification is not sufficient to substantially prevent or impair metabolism. A KBMA bacterium cannot form colonies, for example, as measurable on agar or in vivo in a host cell. To provide a non-limiting example, the genome can be modified with a cross-linking agent, such as psoralen. To provide a non-limiting example, a KBMA bacterium that is not at all impaired in metabolism is one where the genome contains cross-links only in intergenic regions having no regulatory, coding, structural, or biological functions. This is in contrast to a “live” bacterium which can form a colony, although in some embodiments the live bacterium may be attenuated.

“Sample” refers to a sample from a human, animal, placebo, or research sample, e.g., a cell, tissue, organ, fluid, gas, aerosol, slurry, colloid, or coagulated material. “Sample” also refers, e.g., to a cell comprising a fluid or tissue sample or a cell separated from a fluid or tissue sample. “Sample” may also refer to a cell, tissue, organ, or fluid that is freshly taken from a human or animal, or to a cell, tissue, organ, or fluid that is processed or stored.

“Spread” of a bacterium encompasses “cell to cell spread,” that is, transmission of the bacterium from a first host cell to a second host cell, as mediated, in part, by a vesicle. Functions relating to spread include, but are not limited to, e.g., formation of an actin tail, formation of a pseudopod-like extension, and formation of a double-membraned vacuole.

An “effective amount” of a “prime dose” or “prime dosage” refers to the amount of target antigen which elicits a measurable immune response in a mammalian subject as compared to the immune response in the mammalian subject in the absence of administration of the antigen.

A “vaccine” as used herein refers to a composition that is comprised of a heterologous protein, or that is comprised of a nucleic acid encoding the heterologous protein such that when the vaccine is administered to a mammal the heterologous protein is expressed in the mammal for purposes of eliciting an immune response. An immune response may be humoral, cell-mediated, or both.

A “priming vaccine” as used herein refers to a vaccine comprising an agent(s) that are administered to the subject or host in an amount effective to elicit an immune response to an antigen of interest. A priming vaccine may encode the antigen, and may also encode various immune-stimulating cytokines, such as GM-CSF, which act to recruit and activate antigen-presenting cells.

A “boosting vaccine” as used herein refers to a vaccine comprising an agent that encodes an antigen that has an immunologically active portion of the target antigen, and may include the target antigen, be a fragment thereof, and/or be a fusion polypeptide containing at least an immunologically active portion of the target antigen joined to a region that is not normally present in the target antigen. An “effective amount” of a “boost dose” or “boost dosage” refers to the amount of antigen which elicits an immune response to the target antigen upon administration to a mammal which previously has been administered a prime dose of the target antigen.

A “vector set” or “vaccine set” as used herein comprises a priming vector or priming vaccine and a boosting vector or boosting vaccine wherein each encode at least one of a shared immunogenic determinant, a cross reaction immunogenic determinant, a shared antigen, immunogenic protein or peptide, or fragment thereof.

An “antigen” refers to a molecule containing one or more epitopes (either linear, conformational or both) or immunogenic determinants that will stimulate a host's immune-system, such as a mammal's immune system, to make a humoral and/or cellular antigen-specific response. The term is used interchangeably with the term “immunogen.” An antigen may be a whole protein, a truncated protein, a fragment of a protein or a peptide. Antigens may be naturally occurring, genetically engineered variants of the protein, or may be codon optimized for expression in a particular mammalian subject or host. Generally, a B-cell epitope will include at least about 5 amino acids but can be as small as 3-4 amino acids. A T-cell epitope, such as a CTL epitope, will include at least about 7-9 amino acids, and a helper T-cell epitope at least about 12-20 amino acids. Normally, an epitope will include between about 7 and 15 amino acids, such as, 9, 10, 12 or 15 amino acids. The term “antigen” denotes both subunit antigens, (i.e., antigens which are separate and discrete from a whole organism with which the antigen is associated in nature). Antibodies such as anti-idiotype antibodies, or fragments thereof, and synthetic peptide mimotopes, that is synthetic peptides which can mimic an antigen or antigenic determinant, are also captured under the definition of antigen as used herein. For purposes of the present invention, antigens can be from any of several known pathogenic viruses, bacteria, parasites and fungi. They can also be from cancers. Furthermore, for purposes of the present invention, an “antigen” refers to a protein which includes modifications, such as deletions, additions and substitutions, generally conservative in nature, to the naturally occurring sequence, so long as the protein maintains the ability to elicit an immunological response, as defined herein. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the antigens. Antigens of the present invention may also be codon optimized by methods known in the art to improve their expression or immunogenicity in the host. As used herein, a “cross reaction” immunogenic determinant refers to a determinant, epitope, or antigen which is capable of eliciting an immune response to related but not identical antigenic determinants, for example a cross reaction determinant for HIV would be an antigen capable of eliciting an immune response to two or multiple or all members of the HIV antigen across clades.

An “immunological response” or “immune response” to an antigen, or vector or vaccine or composition comprising the antigen, is the development in a mammalian subject of a humoral and/or a cellular immune response to an antigen or antigens present in a vector set. A “cellular immune response” is one mediated by T-lymphocytes and/or other white blood cells, including without limitation NK cells and macrophages. T lymphocytes of the present invention include T cells expressing alpha beta T cell receptor subunits or gamma delta receptor expressing T cells and may be either effector or suppressor T cells. “T lymphocytes” or “T cells” are non-antibody producing lymphocytes that constitute a part of the cell-mediated arm of the immune system. T cells arise from immature lymphocytes that migrate from the bone marrow to the thymus, where they undergo a maturation process under the direction of thymic hormones. Maturing T cells become immunocompetent based on their ability to recognize and bind a specific antigen. Activation of immunocompetent T cells is triggered when an antigen binds to the lymphocyte's surface receptors. It is known that in order to generate T cell responses, antigen must be synthesized within or introduced into cells, subsequently processed into small peptides by the proteasome complex, and translocated into the endoplasmic reticulum/Golgi complex secretory pathway for eventual association with major histocompatibility complex (MHC) class I proteins. Functionally cellular immunity includes antigen specific cytotoxic T cells (CTL). Antigen specific T cells, CTL, or cytotoxic T cells as used herein refers to cells which have specificity for peptide antigens presented in association with proteins encoded by the major histocompatability complex (MHC) or human leukocyte antigens (HLA) as the proteins are referred to in humans. CTLs of the present invention include activated CTL which have become triggered by specific antigen in the context of MHC; and memory CTL or recall CTL to refer to T cells that have become reactivated as a result of re-exposure to antigen as well as cross-reactive CTL. CTLs of the present invention include CD4+ and CD8+ T cells. Activated antigen specific CTLs of the present invention promote the destruction and/or lysis of cells of the subject infected with the pathogen or cancer cell to which the CTL are specific via amongst other things, secretion of chemokines and cytokines including without limitation macrophage inflammatory protein 1a (MIP-1a), MIP-1B, and RANTES; and secretion of soluble factors that suppress the disease state. Cellular immunity of the present invention also refers to antigen specific response produced by the T helper subset of T cells. Helper T cells act to help stimulate the function, and focus the activity of nonspecific effector cells against cells displaying peptide in association with MHC molecules on their surface. A cellular immune response also refers to the production of cytokines, chemokines and other such molecules produced by activated T cells and/or other white blood cells including those derived from CD4 and CD8 T cells and NK cells. A prime dose or boost dose, or a composition or vaccine comprising a prime dose or a boost dose, that elicits a cellular immune response may serve to sensitize a mammalian subject by the presentation of antigen in association with MHC molecules at the cell surface. The cell-mediated immune response is directed at, or near, cells presenting antigen at their surface. In addition, antigen-specific T-lymphocytes can be generated to allow for the future protection of an immunized host. The ability of a particular antigen to stimulate a cell-mediated immunological response may be determined by a number of assays known in the art, such as by lymphoproliferation (lymphocyte activation) assays, CTL cytotoxic cell assays, or by assaying for T-lymphocytes specific for the antigen in a sensitized subject. Such assays are well known in the art. See, e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doe et al., Eur. J. Immunol. (1994) 24:2369-2376. Methods of measuring cell-mediated immune response include measurement of intracellular cytokines or cytokine secretion by T-cell populations, or by measurement of epitope specific T-cells (e.g., by the tetramer technique) (reviewed by McMichael, A. J., and O'Callaghan, C. A., J. Exp. Med. 187(9)1367-1371, 1998; Mcheyzer-Williams, M. G., et al, Immunol. Rev. 150:5-21, 1996; Lalvani, A., et al, J. Exp. Med. 186:859-865, 1997). An immunological response, or immune response, as used herein encompasses one which stimulates the production of CTLs, and/or the production or activation of helper T-cells and/or an antibody-mediated immune response.

An “immunological response” or “immune response” as used herein encompasses at least one or more of the following effects: the production of antibodies by B-cells; and/or the activation of suppressor T-cells and/or T-cells directed specifically to an antigen or antigens present in the vectors, composition or vaccine of interest. In some embodiments, the “immunological response” or “immune response” encompasses the inactivation of suppressor T-cells. As used herein, an “enhanced boost immune response” refers to administration of boost dose by a vaccine that elicits a greater measurable immune response as compared to the response elicited by a single administration of the prime dose.

By “pharmaceutically acceptable” or “pharmacologically acceptable” is meant a material which is not biologically or otherwise undesirable.

As used herein, the term “kit” refers to components packaged and/or marked for use with each other, although not necessarily simultaneously. A kit may contain the priming vaccine and boosting vaccine in separate containers. A kit may also contain the components for a priming vaccine and/or a boosting vaccine in separate containers. A kit may also contain instructions for combining the components so as to formulate an immunogenic composition suitable for administration to a mammal.

A “therapeutic effect” is a lessening of one or more symptoms associated with a disease for which the vaccine(s) are being administered. A “prophylactic effect” is an inhibition of one or more symptoms associated with the disease for which the vaccine(s) are being administered.

As used herein “mammalian subject” or “host” is meant any member of the subphylum chordata, including, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs. The term does not denote a particular age. Thus, both adult and newborn individuals are intended to be covered.

As used herein “radiation therapy” or “radiotherapy” refers to the medical use of ionizing radiation as part of cancer treatment to control malignant cells. Radiotherapy may be used for curative, adjuvant, or palliative treatment. Suitable types of radiotherapy include conventional external beam radiotherapy, stereotactic radiation therapy (e.g., Axesse, Cyberknife, Gamma Knife, Novalis, Primatom, Synergy, X-Knife, TomoTherapy or Trilogy), Intensity-Modulated Radiation Therapy, particle therapy (e.g., proton therapy), brachytherapy, delivery of radioisotopes, etc. This list is not meant to be limiting.

Methods of Eliciting an Immune Response to a Target Antigen

The present invention encompasses methods for eliciting an immune response in a mammal. The target antigens may be those associated with a disease state, for example, those identified as present on a cancerous cell or a pathogenic agent. Subsequent to a primary cancer treatment, a vaccine comprising an attenuated metabolically active Listeria that encodes and expresses an immunologically active portion of the target antigen is administered.

1. Target Antigens

Examples of target antigens that may used in the therapy regimens of the invention are listed in the following table. The target antigen may also be a fragment or fusion polypeptide comprising an immunologically active portion of the antigens listed in the table.

TABLE 1 Antigens. Antigen Reference Tumor antigens Mesothelin GenBank Acc. No. NM_005823; U40434; NM_013404; BC003512 (see also, e.g., Hassan, et al. (2004) Clin. Cancer Res. 10: 3937-3942; Muminova, et al. (2004) BMC Cancer 4: 19; Iacobuzio-Donahue, et al. (2003) Cancer Res. 63: 8614-8622). Wilms' tumor-1 WT-1 isoform A (GenBank Acc. Nos. NM_000378; NP_000369). WT-1 associated protein (Wt-1), isoform B (GenBank Acc. Nos. NM_024424; NP_077742). WT-1 including isoform A; isoform C (GenBank Acc. Nos. NM_024425; NP_077743). WT-1 isoform B; isoform C; isoform D (GenBank Acc. Nos. NM_024426; NP_077744). isoform D. Stratum corneum GenBank Acc. No. NM_005046; NM_139277; AF332583. See also, e.g., chymotryptic enzyme Bondurant, et al. (2005) Clin. Cancer Res. 11: 3446-3454; Santin, et al. (SCCE), and variants (2004) Gynecol. Oncol. 94: 283-288; Shigemasa, et al. (2001) Int. J. thereof. Gynecol. Cancer 11: 454-461; Sepehr, et al. (2001) Oncogene 20: 7368-7374. MHC class I See, e.g., Groh, et al. (2005) Proc. Natl. Acad. Sci. USA 102: chain-related protein A 6461-6466; GenBank Acc. Nos. NM_000247; BC_016929; AY750850; (MICA); MHC class I NM_005931. chain-related protein A (MICB). Gastrin and peptides Harris, et al. (2004) Cancer Res. 64: 5624-5631; Gilliam, et al. derived from gastrin; (2004) Eur. J. Surg. Oncol. 30: 536-543; Laheru and Jaffee (2005) gastrin/CCK-2 receptor Nature Reviews Cancer 5: 459-467. (also known as CCK-B). Glypican-3 (an antigen GenBank Acc. No. NM_004484. Nakatsura, et al. (2003) Biochem. of, e.g., hepatocellular Biophys. Res. Commun. 306: 16-25; Capurro, et al. (2003) carcinoma and Gasteroenterol. 125: 89-97; Nakatsura, et al. (2004) Clin. Cancer melanoma). Res. 10: 6612-6621). Coactosin-like protein. Nakatsura, et al. (2002) Eur. J. Immunol. 32: 826-836; Laheru and Jaffee (2005) Nature Reviews Cancer 5: 459-467. Prostate stem cell antigen GenBank Acc. No. AF043498; AR026974; AR302232 (see also, e.g., (PSCA). Argani, et al. (2001) Cancer Res. 61: 4320-4324; Christiansen, et al. (2003) Prostate 55: 9-19; Fuessel, et al. (2003) 23: 221-228). Prostate acid phosphatase Small, et al. (2000) J. Clin. Oncol. 18: 3894-3903; Altwein and Luboldt (PAP); prostate-specific (1999) Urol. Int. 63: 62-71; Chan, et al. (1999) Prostate 41: 99-109; antigen (PSA); PSM; Ito, et al. (2005) Cancer 103: 242-250; Schmittgen, et al. (2003) Int. PSMA. J. Cancer 107: 323-329; Millon, et al. (1999) Eur. Urol. 36: 278-285. Six-transmembrane See, e.g., Machlenkin, et al. (2005) Cancer Res. 65: 6435-6442; epithelial antigen of GenBank Acc. No. NM_018234; NM_001008410; NM_182915; NM_024636; prostate (STEAP). NM_012449; BC011802. Prostate carcinoma tumor See, e.g., Machlenkin, et al. (2005) Cancer Res. 65: 6435-6442; antigen-1 (PCTA-1). GenBank Acc. No. L78132. Prostate tumor-inducing See, e.g., Machlenkin, et al. (2005) Cancer Res. 65: 6435-6442). gene-1 (PTI-1). Prostate-specific gene See, e.g., Machlenkin, et al. (2005) Cancer Res. 65: 6435-6442). with homology to G protein-coupled receptor. Prostase (an antrogen See, e.g., Machlenkin, et al. (2005) Cancer Res. 65: 6435-6442; regulated serine GenBank Acc. No. BC096178; BC096176; BC096175. protease). Proteinase 3. GenBank Acc. No. X55668. Cancer-testis antigens, GenBank Acc. No. NM_001327 (NY-ESO-1) (see also, e.g., Li, et al. e.g., NY-ESO-1; SCP-1; (2005) Clin. Cancer Res. 11: 1809-1814; Chen, et al. (2004) Proc. Natl. SSX-1; SSX-2; SSX-4; Acad. Sci. USA. 101(25): 9363-9368; Kubuschok, et al. (2004) Int. J. GAGE, CT7; CT8; CT10; Cancer. 109: 568-575; Scanlan, et al. (2004) Cancer Immun. 4: 1; Scanlan, MAGE-1; MAGE-2; et al. (2002) Cancer Res. 62: 4041-4047; Scanlan, et al. (2000) Cancer MAGE-3; MAGE-4; Lett. 150: 155-164; Dalerba, et al. (2001) Int. J. Cancer 93: 85-90; Ries, et MAGE-6; LAGE-1. al. (2005) Int. J. Oncol. 26: 817-824. MAGE-A1, MAGE-A2; Otte, et al. (2001) Cancer Res. 61: 6682-6687; Lee, et al. (2003) Proc. Natl. MAGE-A3; MAGE-A4; Acad. Sci. USA 100: 2651-2656; Sarcevic, et al. (2003) Oncology 64: 443- MAGE-A6; MAGE-A9; 449; Lin, et al. (2004) Clin. Cancer Res. 10: 5708-5716. MAGE-A10; MAGE-A12; GAGE-3/6; NT-SAR-35; BAGE; CA125. GAGE-1; GAGE-2; De Backer, et al. (1999) Cancer Res. 59: 3157-3165; Scarcella, et al. GAGE-3; GAGE-4; (1999) Clin. Cancer Res. 5: 335-341. GAGE-5; GAGE-6; GAGE-7; GAGE-8; GAGE-65; GAGE-11; GAGE-13; GAGE-7B. HIP1R; LMNA; Scanlan, et al. (2002) Cancer Res. 62: 4041-4047. KIAA1416; Seb4D; KNSL6; TRIP4; MBD2; HCAC5; MAGEA3. DAM family of genes, Fleishhauer, et al. (1998) Cancer Res. 58: 2969-2972. e.g., DAM-1; DAM-6. RCAS1. Enjoji, et al. (2004) Dig. Dis. Sci. 49: 1654-1656. RU2. Van Den Eynde, et al. (1999) J. Exp. Med. 190: 1793-1800. CAMEL. Slager, et al. (2004) J. Immunol. 172: 5095-5102; Slager, et al. (2004) Cancer Gene Ther. 11: 227-236. Colon cancer associated Scanlan, et al. (2002) Cancer Res. 62: 4041-4047. antigens, e.g., NY-CO-8; NY-CO-9; NY-CO-13; NY-CO-16; NY-CO-20; NY-CO-38; NY-CO-45; NY-CO-9/HDAC5; NY-CO-41/MBD2; NY-CO-42/TRIP4; NY-CO-95/KIAA1416; KNSL6; seb4D. N-Acetylglucosaminyl- Dosaka-Akita, et al. (2004) Clin. Cancer Res. 10: 1773-1779. tranferase V (GnT-V). Elongation factor 2 Renkvist, et al. (2001) Cancer Immunol Immunother. 50: 3-15. mutated (ELF2M). HOM-MEL-40/SSX2 Neumann, et al. (2004) Int. J. Cancer 112: 661-668; Scanlan, et al. (2000) Cancer Lett. 150: 155-164. BRDT. Scanlan, et al. (2000) Cancer Lett. 150: 155-164. SAGE; HAGE. Sasaki, et al. (2003) Eur. J. Surg. Oncol. 29: 900-903. RAGE. See, e.g., Li, et al. (2004) Am. J. Pathol. 164: 1389-1397; Shirasawa, et al. (2004) Genes to Cells 9: 165-174. MUM-1 (melanoma Gueguen, et al. (1998) J. Immunol. 160: 6188-6194; Hirose, et al. (2005) ubiquitous mutated); Int. J. Hematol. 81: 48-57; Baurain, et al. (2000) J. Immunol. 164: 6057- MUM-2; MUM-2 Arg- 6066; Chiari, et al. (1999) Cancer Res. 59: 5785-5792. Gly mutation; MUM-3. LDLR/FUT fusion Wang, et al. (1999) J. Exp. Med. 189: 1659-1667. protein antigen of melanoma. NY-REN series of renal Scanlan, et al. (2002) Cancer Res. 62: 4041-4047; cancer antigens. Scanlan, et al. (1999) Cancer Res. 83: 456-464. NY-BR series of breast Scanlan, et al. (2002) Cancer Res. 62: 4041-4047; cancer antigens, e.g., Scanlan, et al. (2001) Cancer Immunity 1: 4. NY-BR-62; NY-BR-75; NY-BR-85; NY-BR-62; NY-BR-85. BRCA-1; BRCA-2. Stolier, et al. (2004) Breast J. 10: 475-480; Nicoletto, et al. (2001) Cancer Treat Rev. 27: 295-304. DEK/CAN fusion Von Lindern, et al. (1992) Mol. Cell. Biol. 12: 1687-1697. protein. Ras, e.g., wild type ras, GenBank Acc. Nos. P01112; P01116; M54969; M54968; P01111; ras with mutations at P01112; K00654. See also, e.g., GenBank Acc. Nos. M26261; M34904; codon 12, 13, 59, or 61, K01519; K01520; BC006499; NM_006270; NM_002890; NM_004985; e.g., mutations G12C; NM_033360; NM_176795; NM_005343. G12D; G12R; G12S; G12V; G13D; A59T; Q61H. K-RAS; H-RAS; N-RAS. BRAF (an isoform of Tannapfel, et al. (2005) Am. J. Clin. Pathol. 123: 256-2601; RAF). Tsao and Sober (2005) Dermatol. Clin. 23: 323-333. Melanoma antigens, GenBank Acc. No. NM_206956; NM_206955; NM_206954; including HST-2 NM_206953; NM_006115; NM_005367; NM_004988; AY148486; melanoma cell antigens. U10340; U10339; M77481. See, eg., Suzuki, et al. (1999) J. Immunol. 163: 2783-2791. Survivin GenBank Acc. No. AB028869; U75285 (see also, e.g., Tsuruma, et al. (2004) J. Translational Med. 2: 19 (11 pages); Pisarev, et al. (2003) Clin. Cancer Res. 9: 6523-6533; Siegel, et al. (2003) Br. J. Haematol. 122: 911- 914; Andersen, et al. (2002) Histol. Histopathol. 17: 669-675). MDM-2 NM_002392; NM_006878 (see also, e.g., Mayo, et al. (1997) Cancer Res. 57: 5013-5016; Demidenko and Blagosklonny (2004) Cancer Res. 64: 3653-3660). Methyl-CpG-binding Muller, et al. (2003) Br. J. Cancer 89: 1934-1939; Fang, et al. proteins (MeCP2; (2004) World J. Gastreenterol. 10: 3394-3398. MBD2). NA88-A. Moreau-Aubry, et al. (2000) J. Exp. Med. 191: 1617-1624. Histone deacetylases Waltregny, et al. (2004) Eur. J. Histochem. 48: 273-290; (HDAC), e.g., HDAC5. Scanlan, et al. (2002) Cancer Res. 62: 4041-4047. Cyclophilin B (Cyp-B). Tamura, et al. (2001) Jpn. J. Cancer Res. 92: 762-767. CA 15-3; CA 27.29. Clinton, et al. (2003) Biomed. Sci. Instrum. 39: 408-414. Heat shock protein Faure, et al. (2004) Int. J. Cancer 108: 863-870. Hsp70. GAGE/PAGE family, Brinkmann, et al. (1999) Cancer Res. 59: 1445-1448. e.g., PAGE-1; PAGE-2; PAGE-3; PAGE-4; XAGE-1; XAGE-2; XAGE-3. MAGE-A, B, C, and D Lucas, et al. (2000) Int. J. Cancer 87: 55-60; families. MAGE-B5; Scanlan, et al. (2001) Cancer Immun. 1: 4. MAGE-B6; MAGE-C2; MAGE-C3; MAGE-3; MAGE-6. Kinesin 2; TATA element Scanlan, et al. (2001) Cancer Immun. 30: 1-4. modulatory factor 1; tumor protein D53; NY Alpha-fetoprotein (AFP) Grimm, et al. (2000) Gastroenterol. 119: 1104-1112. SART1; SART2; Kumamuru, et al. (2004) Int. J. Cancer 108: 686-695; Sasatomi, et al. SART3; ART4. (2002) Cancer 94: 1636-1641; Matsumoto, et al. (1998) Jpn. J. Cancer Res. 89: 1292-1295; Tanaka, et al. (2000) Jpn. J. Cancer Res. 91: 1177-1184. Preferentially expressed Matsushita, et al. (2003) Leuk. Lymphoma 44: 439-444; antigen of melanoma Oberthuer, et al. (2004) Clin. Cancer Res. 10: 4307-4313. (PRAME). Carcinoembryonic GenBank Acc. No. M29540; E03352; X98311; M17303 (see also, e.g., antigen (CEA), CAP1-6D Zaremba (1997) Cancer Res. 57: 4570-4577; Sarobe, et al. (2004) Curr. enhancer agonist peptide. Cancer Drug Targets 4: 443-454; Tsang, et al. (1997) Clin. Cancer Res. 3: 2439-2449; Fong, et al. (2001) Proc. Natl. Acad. Sci. USA 98: 8809- 8814). HER-2/neu. Disis, et al. (2004) J. Clin. Immunol. 24: 571-578; Disis and Cheever (1997) Adv. Cancer Res. 71: 343-371. Cdk4; cdk6; p16 (INK4); Ghazizadeh, et al. (2005) Respiration 72: 68-73; Rb protein. Ericson, et al. (2003) Mol. Cancer Res. 1: 654-664. TEL; AML1; Stams, et al. (2005) Clin. Cancer Res. 11: 2974-2980. TEL/AML1. Telomerase (TERT). Nair, et al. (2000) Nat. Med. 6: 1011-1017. 707-AP. Takahashi, et al. (1997) Clin. Cancer Res. 3: 1363-1370. Annexin, e.g., Zimmerman, et al. (2004) Virchows Arch. 445: 368-374. Annexin II. BCR/ABL; BCR/ABL Cobaldda, et al. (2000) Blood 95: 1007-1013; Hakansson, et al. (2004) p210; BCR/ABL p190; Leukemia 18: 538-547; Schwartz, et al. (2003) Semin. Hematol. 40: 87-96; CML-66; CML-28. Lim, et al. (1999) Int. J. Mol. Med. 4: 665-667. BCL2; BLC6; Iqbal, et al. (2004) Am. J. Pathol. 165: 159-166. CD10 protein. CDC27 (this is a Wang, et al. (1999) Science 284: 1351-1354. melanoma antigen). Sperm protein 17 (SP17); Arora, et al. (2005) Mol. Carcinog. 42: 97-108. 14-3-3-zeta; MEMD; KIAA0471; TC21. Tyrosinase-related GenBank Acc. No. NM_001922. (see also, e.g., Bronte, proteins 1 and 2 (TRP-1 et al. (2000) Cancer Res. 60: 253-258). and TRP-2). Gp100/pmel-17. GenBank Acc. Nos. AH003567; U31798; U31799; U31807; U31799 (see also, e.g., Bronte, et al. (2000) Cancer Res. 60: 253-258). TARP. See, e.g., Clifton, et al. (2004) Proc. Natl. Acad. Sci. USA 101: 10166- 10171; Virok, et al. (2005) Infection Immunity 73: 1939-1946. Tyrosinase-related GenBank Acc. No. NM_001922. (see also, e.g., Bronte, et al. (2000) proteins 1 and 2 (TRP-1 Cancer Res. 60: 253-258). and TRP-2). Melanocortin 1 receptor Salazar-Onfray, et al. (1997) Cancer Res. 57: 4348-4355; Reynolds, et al. (MC1R); MAGE-3; (1998) J. Immunol. 161: 6970-6976; Chang, et al. (2002) Clin. Cancer Res. gp100; tyrosinase; 8: 1021-1032. dopachrome tautomerase (TRP-2); MART-1. MUC-1; MUC-2. See, e.g., Davies, et al. (1994) Cancer Lett. 82: 179-184; Gambus, et al. (1995) Int. J. Cancer 60: 146-148; McCool, et al. (1999) Biochem. J. 341: 593-600. Spas-1. U.S. Published patent application No. 20020150588 of Allison, et al. CASP-8; FLICE; MACH. Mandruzzato, et al. (1997) J. Exp. Med. 186: 785-793. CEACAM6; CAP-1. Duxbury, et al. (2004) Biochem. Biophys. Res. Commun. 317: 837-843; Morse, et al. (1999) Clin. Cancer Res. 5: 1331-1338. HMGB1 (a DNA binding Brezniceanu, et al. (2003) FASEB J. 17: 1295-1297. protein and cytokine). ETV6/AML1. Codrington, et al. (2000) Br. J. Haematol. 111: 1071-1079. Mutant and wild type Clements, et al. (2003) Clin. Colorectal Cancer 3: 113-120; Gulmann, et al. forms of adenomatous (2003) Appl. Immunohistochem. Mol. Morphol. 11: 230-237; Jungck, et al. polyposis coli (APC); (2004) Int. J. Colorectal. Dis. 19: 438-445; Wang, et al. (2004) J. Surg. beta-catenin; c-met; p53; Res. 120: 242-248; Abutaily, et al. (2003) J. Pathol. 201: 355-362; Liang, et E-cadherin; al. (2004) Br. J. Surg. 91: 355-361; Shirakawa, et al. (2004) Clin. Cancer cyclooxygenase-2 Res. 10: 4342-4348. (COX-2). Renal cell carcinoma Mulders, et al. (2003) Urol. Clin. North Am. 30: 455-465; Steffens, et al. antigen bound by mAB (1999) Anticancer Res. 19: 1197-1200. G250. EphA2 See. e.g., U.S. Patent Publication No. 2005/0281783 A1; Genbank Accession No. NM_004431 (human); Genbank Accession No. NM_010139 (Mouse); Genbank Accession No. AB038986 (Chicken, partial sequence); GenBank Accession Nos. NP_004422, AAH37166, and AAA53375 (human); GenBank Accession Nos. NP_034269 (mouse), AAH06954 (mouse), XP_345597 (rat), and BAB63910 (chicken). Francisella tularensis antigens Francisella tularensis Complete genome of subspecies Schu S4 (GenBank Acc. No. AJ749949); A and B. of subspecies Schu 4 (GenBank Acc. No. NC_006570). Outer membrane protein (43 kDa) Bevanger, et al. (1988) J. Clin. Microbiol. 27: 922-926; Porsch-Ozcurumez, et al. (2004) Clin. Diagnostic. Lab. Immunol. 11: 1008-1015). Antigenic components of F. tularensis include, e.g., 80 antigens, including 10 kDa and 60 kDa chaperonins (Havlasova, et al. (2002) Proteomics 2: 857-86), nucleoside diphosphate kinase, isocitrate dehydrogenase, RNA-binding protein Hfq, the chaperone ClpB (Havlasova, et al. (2005) Proteomics 5: 2090-2103). See also, e.g., Oyston and Quarry (2005) Antonie Van Leeuwenhoek 87: 277-281; Isherwood, et al. (2005) Adv. Drug Deliv. Rev. 57: 1403-1414; Biagini, et al. (2005) Anal. Bioanal. Chem. 382: 1027-1034. Malarial antigens Circumsporozoite protein See, e.g., Haddad, et al. (2004) Infection Immunity 72: 1594-1602; (CSP); SSP2; HEP17; Hoffman, et al. (1997) Vaccine 15: 842-845; Oliveira-Ferreira and Exp-1 orthologs found in Daniel-Ribeiro (2001) Mem. Inst. Oswaldo Cruz, Rio de Janeiro 96: 221- P. falciparum; and 227. CSP (see, e.g., GenBank Acc. No. AB121024). SSP2 (see, e.g., LSA-1. GenBank Acc. No. AF249739). LSA-1 (see, e.g., GenBank Acc. No. Z30319). Ring-infected erythrocyte See, e.g., Stirnadel, et al. (2000) Int. J. Epidemiol. 29: 579-586; Krzych, et survace protein (RESA); al. (1995) J. Immunol. 155: 4072-4077. See also, Good, et al. (2004) merozoite surface Immunol. Rev. 201: 254-267; Good, et al. (2004) Ann. Rev. Immunol. protein 2 (MSP2); Spf66; 23: 69-99. MSP2 (see, e.g., GenBank Acc. No. X96399; X96397). MSP1 merozoite surface (see, e.g., GenBank Acc. No. X03371). RESA (see, e.g., GenBank Acc. protein 1(MSP1); 195A; No. X05181; X05182). BVp42. Apical membrane See, e.g., Gupta, et al. (2005) Protein Expr. Purif. 41: 186-198. AMA1 antigen 1 (AMA1). (see, e.g., GenBank Acc. No. A′13; AJ494905; AJ490565). Viruses and viral antigens Hepatitis A GenBank Acc. Nos., e.g., NC_001489; AY644670; X83302; K02990; M14707. Hepatitis B Complete genome (see, e.g., GenBank Acc. Nos. AB214516; NC_003977; AB205192; AB205191; AB205190; AJ748098; AB198079; AB198078; AB198076; AB074756). Hepatitis C Complete genome (see, e.g., GenBank Acc. Nos. NC_004102; AJ238800; AJ238799; AJ132997; AJ132996; AJ000009; D84263). Hepatitis D GenBank Acc. Nos, e.g. NC_001653; AB118847; AY261457. Human papillomavirus, See, e.g., Trimble, et al. (2003) Vaccine 21: 4036-4042; Kim, et al. (2004) including all 200+ Gene Ther. 11: 1011-1018; Simon, et al. (2003) Eur. J. Obstet. Gynecol. subtypes (classed in Reprod. Biol. 109: 219-223; Jung, et al. (2004) J. Microbiol. 42: 255- 16 groups), such as the 266; Damasus-Awatai and Freeman-Wang (2003) Curr. Opin. high risk subtypes 16, Obstet. Gynecol. 15: 473-477; Jansen and Shaw (2004) Annu. Rev. 18, 30, 31, 33, 45. Med. 55: 319-331; Roden and Wu (2003) Expert Rev. Vaccines 2: 495-516; de Villiers, et al. (2004) Virology 324: 17-24; Hussain and Paterson (2005) Cancer Immunol. Immunother. 54: 577-586; Molijn, et al. (2005) J. Clin. Virol. 32 (Suppl. 1) S43-S51. GenBank Acc. Nos. AY686584; AY686583; AY686582; NC_006169; NC_006168; NC_006164; NC_001355; NC_001349; NC_005351; NC_001596). Human T-cell See, e.g., Capdepont, et al. (2005) AIDS Res. Hum. Retrovirus lymphotropic virus 21: 28-42; Bhigjee, et al. (1999) AIDS Res. Hum. Restrovirus (HTLV) types I and II, 15: 1229-1233; Vandamme, et al. (1998) J. Virol. 72: 4327-4340; including the Vallejo, et al. (1996) J. Acquir. Immune Defic. Syndr. Hum. HTLV type I subtypes Retrovirol. 13: 384-391. HTLV type I (see, e.g., GenBank Acc. Cosmopolitan, Central Nos. AY563954; AY563953. HTLV type II (see, e.g., GenBank Acc. African, and Nos. L03561; Y13051; AF139382). Austro-Melanesian, and the HTLV type II subtypes Iia, Iib, Iic, and Iid. Coronaviridae, See, e.g., Brian and Baric (2005) Curr. Top. Microbiol. Immunol. including 287: 1-30; Gonzalez, et al. (2003) Arch. Virol. 148: 2207-2235; Coronaviruses, such as Smits, et al. (2003) J. Virol. 77: 9567-9577; Jamieson, et al. (1998) SARS-coronavirus J. Infect. Dis. 178: 1263-1269 (GenBank Acc. Nos. AY348314; (SARS-CoV), and NC_004718; AY394850). Toroviruses. Rubella virus. GenBank Acc. Nos. NC_001545; AF435866. Mumps virus, including See, e.g., Orvell, eta 1. (2002) J. Gen. Virol. 83: 2489-2496. the genotypes A, C, D, See, e.g., GenBank Acc. Nos. AY681495; NC_002200; AY685921; G, H, and I. AF201473. Coxsackie virus A See, e.g., Brown, et al. (2003) J. Virol. 77: 8973-8984. including the serotypes GenBank Acc. Nos. AY421768; AY790926: X67706. 1, 11, 13, 15, 17, 18, 19, 20, 21, 22, and 24 (also known as Human enterovirus C; HEV-C). Coxsackie virus B, See, e.g., Ahn, et al. (2005) J. Med. Virol. 75: 290-294; Patel, et al. including subtypes 1-6. (2004) J. Virol. Methods 120: 167-172; Rezig, et al. (2004) J. Med. Virol. 72: 268-274. GenBank Acc. No. X05690. Human enteroviruses See, e.g., Oberste, et al. (2004) J. Virol. 78: 855-867. Human including, e.g., human enterovirus A (GenBank Acc. Nos. NC_001612); human enterovirus A (HEV-A, enterovirus B (NC_001472); human enterovirus C (NC_001428); CAV2 to CAV8, human enterovirus D (NC_001430). Simian enterovirus A CAV10, CAV12, (GenBank Acc. No. NC_003988). CAV14, CAV16, and EV71) and also including HEV-B (CAV9, CBV1 to CBV6, E1 to E7, E9, E11 to E21, E24 to E27, E29 to E33, and EV69 and E73), as well as HEV. Polioviruses including See, e.g., He, et al. (2003) J. Virol. 77: 4827-4835; Hahsido, et al. PV1, PV2, and PV3. (1999) Microbiol. Immunol. 43: 73-77. GenBank Acc. No. AJ132961 (type 1); AY278550 (type 2); X04468 (type 3). Viral encephalitides See, e.g., Hoke (2005) Mil. Med. 170: 92-105; Estrada-Franco, et al. viruses, including (2004) Emerg. Infect. Dis. 10: 2113-2121; Das, et al. (2004) equine encephalitis, Antiviral Res. 64: 85-92; Aguilar, et al. (2004) Emerg. Infect. Dis. Venezuelan equine 10: 880-888; Weaver, et al. (2004) Arch. Virol. Suppl. 18: 43-64; encephalitis (VEE) Weaver, et al. (2004) Annu. Rev. Entomol. 49: 141-174. Eastern (including subtypes IA, equine encephalitis (GenBank Acc. No. NC_003899; AY722102); IB, IC, ID, IIIC, IIID), Western equine encephalitis (NC_003908). Eastern equine encephalitis (EEE), Western equine encephalitis (WEE), St. Louis encephalitis, Murray Valley (Australian) encephalitis, Japanese encephalitis, and tick-born encephalitis. Human herpesviruses, See, e.g., Studahl, et al. (2000) Scand. J. Infect. Dis. 32: 237-248; including Padilla, et al. (2003) J. Med. Virol. 70 (Suppl. 1) S103-S110; cytomegalovirus Jainkittivong and Langlais (1998) Oral Surg. Oral Med. 85: 399-403. (CMV), Epstein-Barr GenBank Nos. NC_001806 (herpesvirus 1); NC_001798 virus (EBV), human (herpesvirus 2); X04370 and NC_001348 (herpesvirus 3); herpesvirus-1 NC_001345 (herpesvirus 4); NC_001347 (herpesvirus 5); X83413 (HHV-1), HHV-2, and NC_000898 (herpesvirus 6); NC_001716 (herpesvirus 7). HHV-3, HHV-4, Human herpesviruses types 6 and 7 (HHV-6; HHV-7) are disclosed HHV-5, HHV-6, by, e.g., Padilla, et al. (2003) J. Med. Virol. 70 (Suppl. 1)S103- HHV-7, HHV-8, S110. Human herpesvirus 8 (HHV-8), including subtypes A-E, are herpes B virus, herpes disclosed in, e.g., Treurnicht, et al. (2002) J. Med. Virul. 66: 235- simplex virus types 1 240. and 2 (HSV-1, HSV-2), and varicella zoster virus (VZV). HIV-1 including group See, e.g., Smith, et al. (1998) J. Med. Virol. 56: 264-268. See also, M (including subtypes e.g., GenBank Acc. Nos. DQ054367; NC_001802; AY968312; A to J) and group O DQ011180; DQ011179; DQ011178; DQ011177; AY588971; (including any AY588970; AY781127; AY781126; AY970950; AY970949; distinguishable AY970948; X61240; AJ006287; AJ508597; and AJ508596. subtypes) (HIV-2, including subtypes A-E. Epstein-Barr virus See, e.g., Peh, et al. (2002) Pathology 34: 446-450. (EBV), including Epstein-Barr virus strain B95-8 (GenBank Acc. No. V01555). subtypes A and B. Reovirus, including See, e.g., Barthold, et al. (1993) Lab. Anim. Sci. 43: 425-430; Roner, serotypes and strains 1, et al. (1995) Proc. Natl. Acad. Sci. USA 92: 12362-12366; Kedl, et 2, and 3, type 1 Lang, al. (1995) J. Virol. 69: 552-559. GenBank Acc. No. K02739 type 2 Jones, and (sigma-3 gene surface protein). type 3 Dearing. Cytomegalovirus See, e.g., Chern, et al. (1998) J. Infect. Dis. 178: 1149-1153; Vilas (CMV) subtypes Boas, et al. (2003) J. Med. Virol. 71: 404-407; Trincado, et al. include CMV subtypes (2000) J. Med. Virol. 61: 481-487. GenBank Acc. No. X17403. I-VII. Rhinovirus, including Human rhinovirus 2 (GenBank Acc. No. X02316); Human rhinovirus B all serotypes. (GenBank Acc. No. NC_001490); Human rhinovirus 89 (GenBank Acc. No. NC_001617); Human rhinovirus 39 (GenBank Acc. No. AY751783). Adenovirus, including AY803294; NC_004001; AC_000019; AC_000018; AC_000017; all serotypes. AC_000015; AC_000008; AC_000007; AC_000006; AC_000005; AY737798; AY737797; NC_003266; NC_002067; AY594256; AY594254; AY875648; AJ854486; AY163756; AY594255; AY594253; NC_001460; NC_001405; AY598970; AY458656; AY487947; NC_001454; AF534906; AY45969; AY128640; L19443; AY339865; AF532578. Varicella-zoster virus, See, e.g., Loparev, et al. (2004) J. Virol. 78: 8349-8358; including strains and Carr, et al. (2004) J. Med. Virol. 73: 131-136; Takayama genotypes Oka, Dumas, and Takayama (2004) J. Clin. Virol. 29: 113-119. European, Japanese, and Mosaic. Filoviruses, including See, e.g., Geisbert and Jahrling (1995) Virus Res. 39: 129-150; Marburg virus and Hutchinson, et al. (2001) J. Med. Virol. 65: 561-566. Marburg virus Ebola virus, and strains (see, e.g., GenBank Acc. No. NC_001608). Ebola virus (see, e.g., such as Ebola-Sudan GenBank Acc. Nos. NC_006432; AY769362; NC_002549; (EBO-S), Ebola-Zaire AF272001; AF086833). (EBO-Z), and Ebola-Reston (EBO-R). Arenaviruses, including Junin virus, segment S (GenBank Acc. No. NC_005081); Junin virus, lymphocytic segment L (GenBank Acc. No. NC_005080). choriomeningitis (LCM) virus, Lassa virus, Junin virus, and Machupo virus. Rabies virus. See, e.g., GenBank Acc. Nos. NC_001542; AY956319; AY705373; AF499686; AB128149; AB085828; AB009663. Arboviruses, including Dengue virus type 1 (see, e.g., GenBank Acc. Nos. AB195673; West Nile virus, AY762084). Dengue virus type 2 (see, e.g., GenBank Acc. Nos. Dengue viruses 1 to 4, NC_001474; AY702040; AY702039; AY702037). Dengue virus Colorado tick fever type 3 (see, e.g., GenBank Acc. Nos. AY923865; AT858043). virus, Sindbis virus, Dengue virus type 4 (see, e.g., GenBank Acc. Nos. AY947539; Togaviraidae, AY947539; AF326573). Sindbis virus (see, e.g., GenBank Acc. Flaviviridae, Nos. NC_001547; AF429428; J02363; AF103728). West Nile virus Bunyaviridae, (see, e.g., GenBank Acc. Nos. NC_001563; AY603654). Reoviridae, Rhabdoviridae, Orthomyxoviridae, and the like. Poxvirus including Viriola virus (see, e.g., GenBank Acc. Nos. NC_001611; Y16780; orthopoxvirus (variola X72086; X69198). virus, monkeypox virus, vaccinia virus, cowpox virus), yatapoxvirus (tanapox virus, Yaba monkey tumor virus), parapoxvirus, and molluscipoxvirus. Yellow fever. See, e.g., GenBank Acc. No. NC_002031; AY640589; X03700. Hantaviruses, including See, e.g., Elgh, et al. (1997) J. Clin. Microbiol. 35: 1122-1130; serotypes Hantaan Sjolander, et al. (2002) Epidemiol. Infect. 128: 99-103; Zeier, et al. (HTN), Seoul (SEO), (2005) Virus Genes 30: 157-180. GenBank Acc. No. NC_005222 Dobrava (DOB), Sin and NC_005219 (Hantavirus). See also, e.g., GenBank Acc. Nos. Nombre (SN), Puumala NC_005218; NC_005222; NC_005219. (PUU), and Dobrava-like Saaremaa (SAAV). Flaviviruses, including See, e.g., Mukhopadhyay, et al. (2005) Nature Rev. Microbiol. 3: 13- Dengue virus, Japanese 22. GenBank Acc. Nos NC_001474 and AY702040 (Dengue). encephalitis virus, West GenBank Acc. Nos. NC_001563 and AY603654. Nile virus, and yellow fever virus. Measles virus. See, e.g., GenBank Acc. Nos. AB040874 and AY486084. Human Human parainfluenza virus 2 (see, e.g., GenBank Acc. Nos. AB176531; parainfluenzaviruses NC003443). Human parainfluenza virus 3 (see, e.g., GenBank Acc. No. (HPV), including HPV NC_001796). types 1-56. Influenza virus, Influenza nucleocapsid (see, e.g., GenBank Acc. No. AY626145). including influenza Influenza hemagglutinin (see, e.g., GenBank Acc. Nos. AY627885; virus types A, B, AY555153). Influenza neuraminidase (see, e.g., GenBank Acc. Nos. and C. AY555151; AY577316). Influenza matrix protein 2 (see, e.g., GenBank Acc. Nos. AY626144(.Influenza basic protein 1 (see, e.g., GenBank Acc. No. AY627897). Influenza polymerase acid protein (see, e.g., GenBank Acc. No. AY627896). Influenza nucleoprotein (see, e.g., GenBank Acc. Nno. AY627895). Influenza A virus Hemagglutinin of H1N1 (GenBank Acc. No. S67220). Influenza A virus subtypes, e.g., swine matrix protein (GenBank Acc. No. AY700216). Influenza virus A H5H1 viruses (SIV): H1N1 nucleoprotein (GenBank Acc. No. AY646426). H1N1 haemagglutinin influenzaA and swine (GenBank Acc. No. D00837). See also, GenBank Acc. Nos. BD006058; influenza virus. BD006055; BD006052. See also, e.g., Wenrworth, et al. (1994) J. Virol. 68: 2051-2058; Wells, et al. (1991) J.A.M.A. 265: 478-481. Respiratory syncytial Respiratory syncytial virus (RSV) (see, e.g., GenBank Acc. Nos. virus (RSV), including AY353550; NC_001803; NC001781). subgroup A and subgroup B. Rotaviruses, including Human rotavirus C segment 8 (GenBank Acc. No. AJ549087); human rotaviruses A to Human rotavirus G9 strain outer capsid protein (see, e.g., E, bovine rotavirus, GenBank Acc. No. DQ056300); Human rotavirus B strain non-structural rhesus monkey protein 4 (see, e.g., GenBank Acc. No. AY548957); human rotavirus rotavirus, and A strain major inner capsid protein (see, e.g., GenBank Acc. No. human-RVV AY601554). reassortments. Polyomavirus, See, e.g., Engels, et al. (2004) J. Infect. Dis. 190: 2065-2069; including simian Vilchez and Butel (2004) Clin. Microbiol. Rev. 17: 495-508; virus 40 (SV40), JC Shivapurkar, et al. (2004) Cancer Res. 64: 3757-3760; Carbone, et virus (JCV) and BK al. (2003) Oncogene 2: 5173-5180; Barbanti-Brodano, et al. (2004) virus (BKV). Virology 318: 1-9) (SV40 complete genome in, e.g., GenBank Acc. Nos. NC_001669; AF168994; AY271817; AY271816; AY120890; AF345344; AF332562). Coltiviruses, including Attoui, et al. (1998) J. Gen. Virol. 79: 2481-2489. Segments of Colorado tick fever Eyach virus (see, e.g., GenBank Acc. Nos. AF282475; AF282472; virus, Eyach virus. AF282473; AF282478; AF282476; NC_003707; NC_003702; NC_003703; NC_003704; NC_003705; NC_003696; NC_003697; NC_003698; NC_003699; NC_003701; NC_003706; NC_003700; AF282471; AF282477). Calciviruses, including Snow Mountain virus (see, e.g., GenBank Acc. No. AY134748). the genogroups Norwalk, Snow Mountain group (SMA), and Saaporo. Parvoviridae, including See, e.g., Brown (2004) Dev. Biol. (Basel) 118: 71-77; Alvarez-Lafuente, dependovirus, et al. (2005) Ann. Rheum. Dis. 64: 780-782; Ziyaeyan, et al. (2005) Jpn. J. parvovirus (including Infect. Dis. 58: 95-97; Kaufman, et al. (2005) Virology 332: 189-198. parvovirus B19), and erythrovirus.

2. Agents Used in Prime-Boost Approaches

In prime-boost approaches, the adjuvant therapy may comprise administering to the mammal an effective dose of a priming vaccine. The initial vaccine preferably does not contain metabolically active Listeria that encodes the target antigen. Such a vaccine may contain either the target antigen itself, for example, a protein with or without an adjuvant, a tumor cell lysate, an irradiated tumor cell, an antigen-presenting cell pulsed with peptides of the target antigen (e.g. a dendritic cell), or it may contain an agent that provides the target antigen. Suitable agents that provide a target antigen include recombinant vectors, for example, bacteria, viruses, and naked DNA. Recombinant vectors are prepared using standard techniques known in the art, and contain suitable control elements operably linked to the nucleotide sequence encoding the target antigen. See, for example, Plotkin, et al. (eds.) (2003) Vaccines, 4^(th) ed., W.B. Saunders, Co., Phila., Pa.; Sikora, et al. (eds.) (1996) Tumor Immunology Cambridge University Press, Cambridge, UK; Hackett and Ham (eds.) Vaccine Adjuvants, Humana Press, Totowa, N.J.; Isaacson (eds.) (1992) Recombinant DNA Vaccines, Marcel Dekker, NY, N.Y.; Morse, et al. (eds.) (2004) Handbook of Cancer Vaccines, Humana Press, Totowa, N.J.), Liao, et al. (2005) Cancer Res. 65:9089-9098; Dean (2005) Expert Opin. Drug Deliv. 2:227-236; Arlen, et al. (2003) Expert Rev. Vaccines 2:483-493; Dela Cruz, et al. (2003) Vaccine 21:1317-1326; Johansen, et al. (2000) Eur. J. Pharm. Biopharm. 50:413-417; Excler (1998) Vaccine 16:1439-1443; Disis, et al. (1996) J. Immunol. 156:3151-3158). Peptide vaccines are described (see, e.g., McCabe, et al. (1995) Cancer Res. 55:1741-1747; Minev, et al. (1994) Cancer Res. 54:4155-4161; Snyder, et al. (2004) J. Virology 78:7052-7060.

Virus-derived vectors include viruses, modified viruses, and viral particles (see, e.g., Table 2). The virus-derived vectors can be administered directly to a mammalian subject, or can be introduced ex vivo into an antigen presenting cell (APC), where the APC is then administered to the subject.

Viral vectors may be based on, e.g., Togaviruses, including alphaviruses and flaviviruses; alphaviruses, such as Sindbis virus, Sindbis strain SAAR86, Semliki Forest virus (SFV), Venezuelan equine encephalitis (VEE), Eastern equine encephalitis (EEE), Western equine encephalitis, Ross River virus, Sagiyami virus, O'Nyong-nyong virus, Highlands J virus. Flaviviruses, such as Yellow fever virus, Yellow fever strain 17D, Japanese encephalitis, St. Louis encephalitis, Tick-borne encephalitis, Dengue virus, West Nile virus, Kunjin virus (subtype of West Nile virus); arterivirus such as equine arteritis virus; and rubivirus such as rubella virus, herpesvirus, modified vaccinia Ankara (MVA); avipox viral vector; fowlpox vector; vaccinia virus vector; influenza virus vector; adenoviral vector, human papilloma virus vector; bovine papilloma virus vector, and so on. Viral vectors may be based on an orthopoxvirus such as variola virus (smallpox), vaccinia virus (vaccine for smallpox), Ankara (MVA), or Copenhagen strain, camelpox, monkeypox, or cowpox. Viral vectors may be based on an avipoxvirus virus, such as fowlpox virus or canarypox virus.

Adenoviral vectors and adeno-associated virus vectors (AAV) are available, where adenoviral vectors include adenovirus serotype 5 (adeno5; Ad5), adeno6, adeno11, and adeno35. Available are at least 51 human adenovirus serotypes, classified into six subgroups (subgroups A, B, C, D, E, and F). Adenovirus proteins useful, for example, in assessing immune response to an “empty” advenoviral vector, include hexon protein, such as hexon 3 protein, fiber protein, and penton base proteins, and human immune responses to adenoviral proteins have been described (see, e.g., Wu, et al. (2002) J. Virol. 76:12775-12782; Mascola (2006) Nature 441:161-162; Roberts, et al. (2006) Nature 441:239-243).

TABLE 2 Virus-derived vaccine vectors. Adenoviral vectors and Polo and Dubensky (2002) Drug Discovery Today 7: 719-727; Xin, et adeno-associated virus al. (2005) Gene Ther. 12: 1769-1777; Morenweiser (2005) Gene Ther. vectors (AAV). 12 (Suppl. 1) S103-S110; Casimiro, et al. (2005) J. Virol. 79: 15547- 15555; Ferreira, et al. (2005) Gene Ther. 12 Suppl. 1: S73-S83; Baez-Astua, et al. (2005) J. Virol. 79: 12807-12817; Vanniasinkam and Ertl (2005) Curr. Gene Ther. 5: 203-212; Tatsis and Ertl (2004) Mol. Ther. 10: 616-629; Santosuosso, et al. (2005) Viral Immunol. 18: 283-291; Zhou, et al. (1996) J. Virol. 70: 7030-7038; Zhou, et al. (2002) J. Gene Med. 4: 498-509. Vaccinia virus Kim, et al. (2005) Hum. Gen. Ther. 16: 26-34; Kaufman, et al. (2005) J. Clin. Invest. 115: 1903-1912; Kaufman, et al. (2004) J. Clin. Oncol. 22: 2122-2132; Marshall, et al. (2005) J. Clin. Invest. 23: 720-731; Hwang and Sanda (1999) Curr. Opin. Mol. Ther. 1: 471-479; Baldwin, et al. (2003) Clin. Cancer Res. 9: 5205-5213; Modified vaccinia Mackova, et al. (2006) Cancer Immunol. Immunother. 55: 39-46; Ankara (MVA) Meyer, et al. (2005) Cancer Immunol. Immunother. 54: 453-467; Palmowski, et al. (2002) J. Immunol. 168: 4391-4398; Vaccinia derivative Paoletti (1996) Proc. Natl. Acad. Sci. USA 93: 11349-11353; NYVAC Poxviruses, including Kaufman (2005) J. Clin. Oncol. 23: 659-661; Kudo-Saito, et al. avipox, e.g., fowlpox (2004) Clin. Cancer Res. 10: 1090-1099; Greiner, et al. (2002) Cancer and canarypox Res. 62: 6944-6951; Marshall, et al. (2005) J. Clin. Invest. 23: 720- 731; Hwang and Sanda (1999) Curr. Opin. Mol. Ther. 1: 471-479; Hodge, et al. (1997) Vaccine 15: 759-768; Skinner, et al. (2005) Expert Rev. Vaccines 4: 63-76; Rosenberg, et al. (2003) Clin. Cancer Res. 9: 2973-2980. Antigen presenting Di Nicola, et al. (2004) Clin. Cancer Res. 10: 5381-5390; cells transduced with a virus-derived vector. Alphavirus-derived Polo and Dubensky (2002) Drug Discovery Today 7: 719-727; Polo, vectors, e.g., Sindbis et al. (1999) Proc. Natl. Acad. Sci. USA 96: 4598-4603; Schlesinger virus, Semliki Forest (2001) Expert Opin. Biol. Ther. 1: 177-191; Pan, et al. (2005) Proc. virus, and Venezuelan Natl. Acad. Sci. USA 102: 11581-11588; Lundstrom (2003) Expert equine encephalitis Rev. Vaccines 2: 447-459; Shafferman, et al. (1996) Adv. Exp. Med. (VEE). Biol. 397: 41-47; Yamanaka (2004) Int. J. Oncol. 24: 919-923; Atkins, et al. (2004) Curr. Cancer Drug Targets 4: 597-607. Chimeric virus-derived Sindbis virus/Venezualan equine encephalitis virus (SINV/VEEV) vectors, such as (see, e.g., Perri, et al. (2003) J. Virol. 77: 10394-10403; Paessler, et al. chimeric alphaviruses. (2003) J. Virol. 77: 9278-9286). Herpesviruses, Hellebrand, et al. (2006) Gene Ther. 13: 150-162; Lauterbach, et al. including herpes (2005) J. Gen. Virol. 86: 2401-2410; Zibert, et al. (2005) Gene Ther. simplex and 12: 1707-1717; Thiry, et al. (2006) Vet. Microbiol. 113: 171-177; Epstein-Barr Trapp, et al. (2005) J. Virol. 79: 5445-5454. virus-derived vectors Rhinoviruses Dollenmaier, et al. (2001) Virology 281: 216-230; Arnold, et al. (1996) Intervirology 39: 72-78. Lentiviruses DePolo, et al. (2000) Mol. Ther. 2: 218-222; Pellinen, et al. (2004) Int. J. Oncol. 25: 1753-1762; Esslinger, et al. (2003) J. Clin. Invest. 111: 1673-1681; Kikuchi, et al. (2004) Clin. Cancer Res. 10: 1835- 1842; Kim, et al. (2005) Hum. Gene Ther. 16: 1255-1266. Viral particle vaccines Polo and Dubensky (2002) Drug Discovery Today 7: 719-727; Cheng, et al. (2002) Hum. Gene Ther. 13: 553-568; Lin, et al. (2003) Mol. Ther. 8: 559-566; Balasuriya, et al. (2000) J. Virol. 74: 10623-10630; Goldberg, et al. (2005) Clin. Cancer Res. 11: 8114-8121; Johnston, et al. (2005) Vaccine 23: 4969-4979; Quinnan, et al. (2005) J. Virol. 79: 3358-3369; Cassetti, et al. (2004) Vaccine 22: 520-527; Williamson, et al. (2003) AIDS Res. Hum. Retroviruses 19: 133-144; Perri, et al. (2003) J. Virol. 77: 10394-10403; Da Silva, et al. (2003) Vaccine 21: 3219-3227;

Antigen presenting cell (APC) vectors, such as a dendritic cell (DC) vector, include cells that are loaded with an antigen, loaded with a tumor lysate, or transfected with a composition comprising a nucleic acid, where the nucleic acid can be, e.g., a plasmid, mRNA, or virus. DC/tumor fusion vaccines may also be used. See, e.g., Di Nicola, et al. (2004) Clin. Cancer Res. 10:5381-5390; Cerundolo, et al. (2004) Nature Immunol. 5:7-10; Parmiani, et al. (2002) J. Natl. Cancer Inst. 94:805-818; Kao, et al. (2005) Immunol. Lett. 101:154-159; Geiger, et al. (2005) J. Transl. Med. 3:29; Osada, et al. (2005) Cancer Immunol. Immunother. November 5, 1-10 [epub ahead of print]; Malowany, et al. (2005) Mol. Ther. 13:766-775; Morse and Lyerly (2002) World J. Surg. 26:819-825; Gabrilovich (2002) Curr. Opin. Mol. Ther. 4:454-458; Morse, et al. (2003) Clin. Breast Cancer 3 Supp1.4:S164-5172; Morse, et al. (2002) Cancer Chemother. Biol. Response Modif. 20:385-390; Arlen, et al. (2003) Expert Rev. Vaccines 2:483-493; Morse and Lyerly (1998) Expert Opin. Investig. Drugs 7:1617-1627; Hirschowitz, et al. (2004) J. Clin. Oncol. 22:2808-2815; Vasir, et al. (2005) Br. J. Haematol. 129:687-700; Koido, et al. (2005) Gynecol. Oncol. 99:462-471.

Tumor cells, for example, autologous and allogeneic tumor cells, are available as vaccines (Arlen, et al. (2005) Semin. Oncol. 32:549-555). A vaccine may also comprise a modified tumor cell, for example, a tumor cell lysate, or an irradiated tumor cell. The tumor cell can also be modified by incorporating a nucleic acid encoding an molecule such as a cytokine (GM-CSF, IL-12, IL-15, and the like), a NKG2D ligand, CD40L, CD80, CD86, and the like (see, e.g., Dranoff (2002) Immunol. Rev. 188:147-154; Jain, et al. (2003) Ann. Surg. Oncol. 10:810-820; Borrello and Pardoll (2002) Cytokine Growth Factor Rev. 13:185-193; Chen, et al. (2005) Cancer Immunol. Immunother. 27:1-11; Kjaergaard, et al. (2005) J. Neurosurg. 103:156-164; Tai, et al. (2004) J. Biomed. Sci. 11:228-238; Schwaab, et al. (2004) J. Urol. 171:1036-1042; Friese, et al. (2003) Cancer Res. 63:8996-9006; Briones, et al. (2002) Cancer Res. 62:3195-3199; Vieweg and Dannull (2003) Urol. Clin. North Am. 30:633-643; Mincheff, et al. (2001) Crit. Rev. Oncol. Hematol. 39:125-132).

Vaccines may include naked DNA vectors and naked RNA vectors. These vaccines containing nucleic acids may be administered by a gene gun, electroporation, bacterial ghosts, microspheres, microparticles, liposomes, polycationic nanoparticles, and the like (see, e.g., Donnelly, et al. (1997) Ann. Rev. Immunol. 15:617-648; Mincheff, et al. (2001) Crit. Rev. Oncol. Hematol. 39:125-132; Song, et al. (2005) J. Virol. 79:9854-9861; Estcourt, et al. (2004) Immunol. Rev. 199:144-155).

Reagents and methodologies for administration of naked nucleic acids, e.g., by way of a gene gun, intradermic, intramuscular, and electroporation methods, are available. The nucleic acid vaccines may comprise a locked nucleic acid (LNA), where the LNA allows for attachment of a functional moiety to the plasmid DNA, and where the functional moiety can be an adjuvant (see, e.g., Fensterle, et al. (1999) J. Immunol. 163:4510-4518; Strugnell, et al. (1997) Immunol. Cell Biol. 75:364-369; Hertoughs, et al. (2003) Nucleic Acids Res. 31:5817-5830; Trimble, et al. (2003) Vaccine 21:4036-4042; Nishitani, et al. (2000) Mol. Urol. 4:47-50; Tuting (1999) Curr. Opin. Mol. Ther. 1:216-225). Nucleic acid vaccines can be used in combination with reagents that promote migration of immature dendritic cells towards the vaccine, and a reagent that promotes migration of mature DCs to the draining lymph node where priming can occur, where these reagents encompass MIP-1alpha and Flt3L (see, e.g., Kutzler and Weiner (2004) J. Clin. Invest. 114:1241-1244; Sumida, et al. (2004) J. Clin. Invest. 114:1334-1342).

Bacterial vectors include, for example, Salmonella, Shigella, Yersinia, Lactobacillus, Streptococcus, Bacille Calmette-Guerin, Bacillus anthracis, and Escherichia coli. The bacterium can be engineered to contain a nucleic acid encoding a recombinant antigen, a heterologous antigen, or an antigen derived from a tumor, cancer cell, or infective agent. Moreover, the bacterium can modified to be attenuated. In another aspect, the non-listerial bacterial vaccine can be absent of any nucleic acid encoding a recombinant antigen (see, e.g., Xu, et al. (2003) Vaccine 21:644-648; Pasetti, et al. (2003) J. Virol. 77:5209-5219; Loessner and Weiss (2004) Expert Opin. Biol. Ther. 4:157-168; Grangette, et al. (2002) Vaccine 20:3304-3309; Byrd, et al. (2002) Vaccine 20:2197-2205; Edelman, et al. (1999) Vaccine 17:904-914; Domenech, et al. (2005) Microbes and Infection 7:860-866).

Killed but metabolically active (“KBMA”) bacteria, and particularly KBMA Listeria can be prepared from live bacteria by treatment with a DNA cross-linking agent (e.g., psoralen) and/or by inactivating at least one gene that mediates DNA repair, e.g., a recombinational repair gene (e.g., recA) or an ultraviolet light damage repair gene (e.g., uvrA, uvrB, uvrAB, uvrC, uvrD, phrA, phrB) (see, e.g., U.S. Pat. Publ. Nos. 2004/0228877 and 2004/0197343 of Dubensky, et al., each of which is hereby incorporated by reference herein in its entirety).

One type of KBMA Listeria are Listeria uvrAB engineered to express a heterologous antigen, where the engineered bacterium is treated with a nucleic acid cross-linking agent, a psoralen compound, a nitrogen mustard compound, 4′-(4-amino-2-oxa)butyl-4,5′,8-trimethylpsoralen, or beta-alanine, N-(acridine-9-yl), 2-[bis(2-chloroethyl)amino]ethyl ester. See also, e.g., U.S. Publ. Pat. Appl. No. US 2004/0197343, MODIFIED FREE-LIVING MICROBES, VACCINE COMPOSITIONS AND METHODS OF USE THEREOF, of Dubensky, et al.; Brockstedt, et al (2005) Nature Med. 11:853-860).

In some embodiments, the priming vaccine comprises an agent selected from the group consisting of a vaccinia virus (VV) vector, a dendritic cell (DC) vector, an adenoviral vector, a naked DNA vector, and GVAX® (CELL GENESYS, INC.).

3. Listeria Used in Adjuvant Therapy

In certain embodiments, the invention includes the use of a Listeria bacterium, where the Listeria is attenuated. The attenuation can result from mutating one or more genes encoding a virulence factor, such as actA, internalin B (inlB), p60 (autolysin), listeriolysin O (LLO; hly gene), phosphatidylcholine phospholipase C (PC-PLC), phosphatidylinositol-specific phospholipase C (PI-PLC; plcA gene), lipoate protein ligase, and well as genes disclosed in ENGINEERED LISTERIA AND METHODS OF USE THEREOF, U.S. Ser. No. 11/395,197 (filed Mar. 30, 2006), assigned to Cerus Corporation. The methods of the invention include, but are not limited to, use of one or more of the listerial species and strains identified therein. For example, the invention encompasses the use of a Listeria bacterium that is, or is derived from, Listeria monocytogenes. Also useful are other species of Listeria, such as L. innocua that are engineered to express one or more of listeriolysin O (hly gene; LLO), plcA, plcB, or other genes such as a virulence gene or gene mediating entry into a host cell (see, e.g., Johnson, et al. (2004) Appl. Environ. Microbiol. 70:4256-4266; Slaghuis, et al. (2004) J. Infect. Dis. 189:393-401; Milohanic, et al. (2003) Mol. Microbiol. 47:1613-1625). The attenuated strains of Listeria suitable for use in the boost vaccines of the invention may be prepared as described in PCT/US2004/003429 and in PCT/US2004/044080. All of the above applications are incorporated by reference herein in their entirety.

Nonlimiting examples of attenuated Listeria are described, e.g., in the following patent publications, each of which is hereby incorporated by reference herein in its entirety: U.S. Patent Publication No. 2004/0228877; U.S. Patent Publication No. 2004/0197343; and U.S. Patent Publication No. 2005/0249748. Nonlimiting examples are also provided, e.g., in U.S. patent application Ser. No. 11/395,197, filed Mar. 30, 2006, which is hereby incorporated by reference herein in its entirety.

4. Vaccine Compositions

In addition to the agents described above, the vaccine compositions of the invention may further comprise various excipients, adjuvants, carriers, auxiliary substances, modulating agents, and the like. A carrier, which is optionally present, is a molecule that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycollic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Examples of particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) and poly(lactide-co-glycolides), known as PLG. See, e.g., Jeffery et al., Pharm. Res. (1993) 10:362-368; McGee J P, et al., J Microencapsul. 14(2):197-210, 1997; O'Hagan D T, et al., Vaccine 11(2):149-54, 1993. Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents (“adjuvants”). Furthermore, the antigen may be conjugated to a bacterial toxoid, such as toxoid from diphtheria, tetanus, cholera, etc., as well as toxins derived from E. coli. Such adjuvants include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59 (International Publication No. WO 90/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and MDP either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi™ adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detoxu); (3) saponin adjuvants, such as Stimulon™ (Cambridge Bioscience, Worcester, Mass.) may be used or particle generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins (IL-1, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), beta chemokines (MIP, 1-alpha, 1-beta Rantes, etc.); (6) detoxified mutants of a bacterial ADP-ribosylating toxin such as a cholera toxin (CT), a pertussis toxin (PT), or an E. coli heat-labile toxin (LT), particularly LT-K63 (where lysine is substituted for the wild-type amino acid at position 63) LT-R72 (where arginine is substituted for the wild-type amino acid at position 72), CT-S 109 (where serine is substituted for the wild-type amino acid at position 109), and PT-K9/G129 (where lysine is substituted for the wild-type amino acid at position 9 and glycine substituted at position 129) (see, e.g., International Publication Nos. WO93/13202 and WO92/19265); and (7) other substances that act as immunostimulating agents to enhance the effectiveness of the composition.

5. Preparation of Vaccine Compositions

The vaccines are prepared by methods known to those of skill in the art. Usually, one or more of the agents described above (for use in prime or boost vaccines) are prepared by mixing a desired amount of the agent with a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients include, but are not limited to, sterile distilled water, saline, phosphate buffered solutions, amino acid-based buffers, or bicarbonate buffered solutions.

6. Vaccine Administration

An effective amount of a priming vector or boosting vector to be supplied in one or multiple doses of a vaccine can be determined by one of skill in the art. Such an amount will fall in a range that can be determined through routine trials.

The prime vaccine and the boost vaccine can be administered by any one or combination of the following routes. In one aspect, the prime vaccine and boost vaccine are administered by the same route. In another aspect, the prime vaccine and boost vaccine are administered by different routes. The term “different routes” encompasses, but is not limited to, different sites on the body, for example, a site that is oral, non-oral, enteral, parenteral, rectal, intranode (lymph node), intravenous, arterial, subcutaneous, intramuscular, intratumor, peritumor, intratumor, infusion, mucosal, nasal, in the cerebrospinal space or cerebrospinal fluid, and so on, as well as by different modes, for example, oral, intravenous, and intramuscular.

An effective amount of a prime or boost vaccine may be given in one dose, but is not restricted to one dose. Thus, the administration can be two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more, administrations of the vaccine. Where there is more than one administration of a vaccine the administrations can be spaced by time intervals of one minute, two minutes, three, four, five, six, seven, eight, nine, ten, or more minutes, by intervals of about one hour, two hours, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, and so on. In the context of hours, the term “about” means plus or minus any time interval within 30 minutes. The administrations can also be spaced by time intervals of one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, and combinations thereof. The invention is not limited to dosing intervals that are spaced equally in time, but encompass doses at non-equal intervals, such as a priming schedule consisting of administration at 1 day, 4 days, 7 days, and 25 days, just to provide a non-limiting example.

The following may be taken into consideration in determining the relative timing of the prime vaccine and boost vaccine. It has been found that administration of an antigen, or nucleic acid encoding an antigen, can stimulate expansion of antigen-specific immune cells, resulting in a peak, followed by contraction of the number of antigen specific immune cells (see, e.g., Badovinac, et al. (2002) Nature Immunol. 3:619-626). Initiation of the boost vaccination can be administered before the peak is reached, coincident with the peak, or after the peak.

Administration of the boost vaccination can be initiated when a population of antigen-specific immune cells has expanded (increased in number) to at least 20% the maximal number of antigen-specific immune cells that is eventually attained; to at least 30%; to at least 40%; to at least 50%; to at least 60%; to at least 70%; to at least 80%; to at least 90%; to at least 95%; to at least 99% the maximal number of antigen-specific immune cells that is eventually attained. Additional schedules of prime-boost vaccines are available, for example, the boost vaccination can be initiated when the population of antigen-specific cells has contracted to under 90% the maximal number of antigen-specific cells; under 80%; under 70%; under 60%; under 50%; under 40%; under 30%; under 20%; under 10%; under 5%; under 1.0%; under 0.5%; under 0.1%; under 0.05%; or under 0.01% the maximal number of antigen-specific immune cells. The antigen-specific cells can be identified as specific for a vector-specific antigen (specific for empty vector), or specific for a heterologous antigen expressed by a nucleic acid contained in the vector.

In other aspects, administration of the boost vaccination can be initiated at about 5 days after the prime vaccination is initiated; about 10 days after the prime vaccination is initiated; about 15 days; about 20 days; about 25 days; about 30 days; about 35 days; about 40 days; about 45 days; about 50 days; about 55 days; about 60 days; about 65 days; about 70 days; about 75 days; about 80 days, about 6 months, and about 1 year after administration of the prime vaccination is initiated.

The boost vaccination can be administered 5-10 days after the prime vaccination; 10-15 days after the prime vaccination; 15-20 days after the prime vaccination; 20-25 days after the prime vaccination; 25-30 days after the prime vaccination; 30-40 days after the prime vaccination; 40-50 days after the prime vaccination; 50-60 days after the prime vaccination; 60-70 days after the prime vaccination; and so on.

The period of time between initiation of the prime vaccination and initiating the boost vaccination can be determined by one of skill in the art. For example, it can be based on an algorithm that is sensitive to physiologic parameters measured after the prime immunization has occurred.

The dosage and regimen will be determined, at least in part, be determined by the potency of the modality, the vaccine delivery employed, the need of the subject and be dependent on the judgment of the practitioner.

For example, the Listeria in the vaccines used in the invention can be administered in a dose, or dosages, where each dose comprises between 10⁷ and 10⁸ Listeria per 70 kg body weight; 2×10⁷ and 2×10⁸ Listeria per 70 kg body weight; 5×10⁷ and 5×10⁸ Listeria per 70 kg body weight; 10⁸ and 10⁹ Listeria per 70 kg body weight; between 2.0×10⁸ and 2.0×10⁹ Listeria per 70 kg; between 5.0×10⁸ to 5.0×10⁹ Listeria per 70 kg; between 10⁹ and 10¹⁰ Listeria per 70 kg; between 2×10⁹ and 2×10¹⁰ Listeria per 70 kg; between 5×10⁹ and 5×10¹⁰ Listeria per 70 kg; between 10¹¹ and 10¹² Listeria per 70 kg; between 2×10¹¹ and 2×10¹² Listeria per 70 kg; between 5×10¹¹ and 5×10¹² Listeria per 70 kg; between 10¹² and 10¹³ Listeria per 70 kg; between 2×10¹² and 2×10¹³ Listeria per 70 kg; between 5×10¹² and 5×10¹³ Listeria per 70 kg; and so on, wet weight. Also provided are each of the above doses, based in a per 1.7 square meters surface area basis, or on a 1.5 kg liver weight basis. It is to be noted that a mouse liver, at the time of administering the Listeria of the invention, weighs about 1.5 grams. Human liver weighs about 1.5 kilograms.

In some embodiments of the invention the boost dose of Listeria will enhance the prime dose immune response by at least two-fold, at times between about three- and five-fold or five-fold to ten-fold, or from ten-fold to 100-fold or greater. In some embodiments of the invention the prime dose and boost dose will have a synergistic effect on the immune response. In some embodiments of the invention the enhanced immune response will include a T-cell response, and in some embodiments the T-cell response will be a CD8⁺ T-cell response. In some embodiments of the invention the prime dose and boost dose will break the mammal's tolerogenic state towards the target antigen. Examples of all of these embodiments are provided below.

7. Methods of Measuring Immune Response

A variety of in vitro and in vivo assays are known in the art for measuring an immune response, including measuring humoral and cellular immune responses, which include but are not limited to standard immunoassays, such as RIA, ELISA assays; intracellular staining; T cell assays including for example, lymphoproliferation (lymphocyte activation) assays, CTL cytotoxic cell assays, or by assaying for T-lymphocytes specific for the antigen in a sensitized subject. Such assays are well known in the art. See, e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doe et al., Eur. J. Immunol. (1994) 24:2369-2376. Recent methods of measuring cell-mediated immune response include measurement of intracellular cytokines or cytokine secretion by T-cell populations, or by measurement of epitope specific T-cells (e.g., by the tetramer technique) (reviewed by McMichael, A. J., and O'Callaghan, C.A., J. Exp. Med. 187(9)1367-1371, 1998; Mcheyzer-Williams, M. G., et al., Immunol. Rev. 150:5-21, 1996; Lalvani, A., et al., J. Exp. Med. 186:859-865, 1997). In illustrative embodiments disclosed herein, the enzyme-linked immunospot (ELISPOT) assay is used to detect and analyze individual cells that secrete interferon-γ (IFN-)γ. ELISPOT IFN-γ assays and reagents are available at BD Biosciences 2350 Qume Drive San Jose, Calif., 95131. The ELISPOT assay is capable of detecting cytokine producing cells from both activated naïve and memory T-cell populations and derives its specificity and sensitivity by employing high affinity capture and detection antibodies and enzyme-amplification. Additional information regarding the use of ELISPOT assay is provided in J. Immunol. Methods. 2001, 254(1-2):59. Animal models, e.g. non-human primates, are known in the art. For example, the mouse is an accepted model for human immune response. Mouse NK cell response to tumors is an accepted model for human NK cell response to tumors. Additionally, mouse T cells are a model for human T cells, mouse dendritic cells (DCs) are a model for human DCs, mouse NKT cells are a model for human NKT cells, mouse innate response is an accepted model for human innate response, and so on. Model studies are disclosed, for example, for CD8⁺T cells, central memory T cells, and effector memory T cells (see, e.g., Walzer, et al. (2002) J. Immunol. 168:2704-2711); the two subsets of NK cells (see, e.g., Chakir, et al. (2000) J. Immunol. 165:4985-4993; Smith, et al. (2000) J. Exp. Med. 191:1341-1354; Ehrlich, et al. (2005) J. Immunol. 174:1922-1931; Peritt, et al. (1998) J. Immunol. 161:5821-5824); NKT cells (see, e.g., Couedel, et al. (1998) Eur. J. Immunol. 28:4391-4397; Sakamoto, et al. (1999) J. Allergy Clin. Immunol. 103:S445-S451; Saikh, et al. (2003) J. Infect. Dis. 188:1562-1570; Emoto, et al. (1997) Infection Immunity 65:5003-5009; Taniguchi, et al. (2003) Annu. Rev. Immunol. 21:483-513; Sidobre, et al. (2004) Proc. Natl. Acad. Sci. 101:12254-12259); monocytes/macrophages (Sunderkotter, et al. (2004) J. Immunol. 172:4410-4417); the two lineages of DCs (Boonstra, et al. (2003) J. Exp. Med. 197:101-109; Donnenberg, et al. (2001) Transplantation 72:1946-1951; Becker (2003) Virus Genes 26:119-130; Carine, et al. (2003) J. Immunol. 171:6466-6477; Penna, et al. (2002) J. Immunol. 69:6673-6676; Alferink, et al. (2003) J. Exp. Med. 197:585-599). Mouse innate response, including the Toll-Like Receptors (TLRs), is a model for human innate immune response, as disclosed (see, e.g., Janssens and Beyaert (2003) Clinical Microb. Revs. 16:637-646). Mouse neutrophils are an accepted model for human neutrophils (see, e.g., Kobayashi, et al. (2003) Proc. Natl. Acad. Sci. USA 100:10948-10953; Tones, et al. (2004) 72:2131-2139; Sibelius, et al. (1999) Infection Immunity 67:1125-1130; Tvinnereim, et al. (2004) J. Immunol. 173:1994-2002). Murine immune response to Listeria is an accepted model for human response to Listeria (see, e.g., Kolb-Maurer, et al. (2000) Infection Immunity 68:3680-3688; Brzoza, et al. (2004) J. Immunol. 173:2641-2651).

8. Uses of the Prime-Boost Regimen

Cancers and infections can be treated and/or inhibited by administering reagents that modulate the immune system. The prime-boost methods encompassed within the invention give rise to immune responses that are upregulated, and include breaking tolerance to self-antigens. Thus, it is expected that these prime-boost methods will be useful in inhibiting the growth of cancers and/or ameliorating one or more symptoms associated with a cancer. It is also expected that the prime-boost methods will be useful in the prophylaxis and/or treatment of a disease caused by a pathogenic agent.

In addition to the above, these regimens can be used to determine whether a mammal will be responsive to a treatment. For example, when a prime-boost regimen towards a specific antigen is used, failure to obtain a significant immune response after the boost suggests that the mammal is non-responsive towards the target antigen and an alternative mode of treatment should be pursued. Examples of this could be when the genetic background of the cancer or pathogenic agent is such that the target antigen is absent or modified in a way that it is not cross-reactive with the target antigen.

EXAMPLES

It is to be understood that this invention is not limited to particular examples disclosed herein, as such may vary. It is also to be understood that the examples are not intended to be limiting since the scope of the present invention is delineated by the appended claims.

General Information on Methods Used in the Examples

Immune responses to vaccines were assessed by harvesting splenocytes, a source that provides cells of the immune system, including T cells and dendritic cells (DCs). Antigen-specific immune responses were measured by incubating splenocytes with one or more peptides and measuring immune cell activity, where activity was determined by intracellular staining (ICS) and elispot assays. In some assays, only a single peptide was added, where the peptide contained only one epitope of a tumor antigen. In other assays, an entire library of peptides was added, encompassing the entire length of the antigen.

ICS assays involve permeabilizing the splenocytes, and treating with an antibody that binds cytokines that have accumulated inside the immune cell, where the antibody allows fluorescent tagging. Brefeldin blocks protein transport, and provokes the accumulation of cytokines within the immune cell.

Elispot (enzyme-linked immunospot) assays are sensitive to secreted proteins, where the proteins are secreted over a period of time from immune cells resting in a well. A capture antibody is bound to the well, which immobilized secreted cytokine. After the secretory period, the cells are removed, and a detection antibody is used to detect immobilized cytokine. The capture antibody and detection antibody bind to different regions of the cytokine. Methodological details of the ICS and elispot assays are disclosed (see, e.g., U.S. Pat. Appl. Pub. No. 2005/0249748, published Nov. 10, 2005, of Dubensky, et al.).

Where the administered vector contained a nucleic acid encoding ovalbumin, analysis of any induced immune response by way of ICS assays or elispot assays used a standard peptide from ovalbumin, OVA₂₅₇₋₂₆₄ (SIINFEKL (SEQ ID NO:1)), where the peptide was added to and incubated with the splenocyte preparation.

The nucleic acid sequence of human Mesothelin, used in the following constructs, was that identified in ENGINEERED LISTERIA AND METHODS OF USE THEREOF, U.S. Ser. No. 11/395,197, filed Mar. 30, 2006, assigned to Cerus Corporation. The Mesothelin peptide library, which spanned the entire length of human Mesothelin, consisted of 153 peptides, each a 15-mer, with each 15-mer overlapping the next 15-mer by eleven amino acids.

Lm-hMeso38, a construct of Listeria monocytogenes used in the following examples, is identified in Table 1. See also, ENGINEERED LISTERIA AND METHODS OF USE THEREOF, U.S. Ser. No. 11/395,197, filed Mar. 30, 2006. Lm-mMeso is the same construct as Lm-hMeso38 except that the full length human Mesothelin sequence is substituted with the full length mouse Mesothelin sequence. The full length mouse sequence is available from GenBank Acc. No. NM_(—)018857.

TABLE 3 Listeria monocytogenes-hMeso38 (Lm-hMeso38). Promoter Secretory operably linked sequence in antigen Secretory Strain Genetic Locus of expression sequence (trivial name) Construct background integration cassette (SS) Lm-hMeso38 ActA-N100-hme ΔactΔΔinlB inlB ActA ActA-N100 so [deltaSS]. The ActA-N100 human Mesothelin sequence was deleted in its signal sequence, but was not deleted in its GPI anchor. “ActA-N100” is the first 100 amino acids of ActA, where the first 29 of these amino acids is the signal sequence.

The preparation of Listeria monocytogenes encoding OVA (“Lm-OVA”) was as discussed in U.S. Patent Publication No. 2004/0197343, (U.S. Ser. No. 10/773,618).

The vaccinia virus-derived vector (VV-OVA), which contains a nucleic acid encoding full-length ovalbumin, was prepared and provided by N. P. Restifo, as described (Overwijk, et al. (1998) J. Exp. Med. 188:277-286).

The adenovirus-based vectors contained a nucleic acid encoding full-length human Mesothelin or full-length mouse Mesothelin (Ad-hMeso or Ad-mMeso). Control Ad vectors not encoding a heterologous antigen, otherwise known as “Empty Ad vector” were also used. All control and antigen-encoding Ad vectors were based on adenovirus serotype 5 with E1 and E3 regions deleted, and were derived utilizing the “AdEasy” system obtained from Stratagene (San Diego, Calif.) and derived according to the Methods described by the supplier. Antigens were cloned at the E1 locus. The nucleic acid encoding the heterologous antigen was integrated into the shuttle vector of AdEasy in operable linkage with the CMV promoter.

GVAX® refers to an inactivated tumor cell containing a nucleic acid that encodes mouse granulocyte macrophage-colony stimulation factor (GM-CSF), where the tumor cell line was CT-26 cells, a cell line that expresses the gp70. AH1 is an epitope of gp70, an immunodominant antigen of CT26 cells. GVAX® (CELL GENESYS, INC.) was prepared and administered as disclosed (see, e.g., Yoshimura, et al. (2006) Cancer Res. 66:1096-1104; Jain, et al. (2003) Annals Surgical Oncol. 10:810-820; Zhou, et al. (2005) Cancer Res. 65:1079-1088; Chang, et al. (2000) Int. J. Cancer 86:725-730; Borrello and Pardoll (2002) Cytokine Growth Factor Rev. 13:185-193; Thomas, et al. (1998) Human Gene Ther. 9:835-843).

Killed but metabolically active Lm (“KBMA-Lm”), were prepared by treating live Lm deleted of the uvrAB genes, whose expression in combination with the uvrC gene product forms the exonuclease required for nucleotide excision repair, with psoralen and ultraviolet light, resulting in a small amount of cross-linking of the genome (see, e.g., U.S. Ser. No. 10/773,618. Pub. No. US 2004/0197343. MODIFIED FREE-LIVING MICROBES, VACCINE COMPOSITIONS AND METHODS OF USE THEREOF, of Dubensky, et al.; Brockstedt, et al. (2005) Nature Med. 11:853-860).

Example 1

C57BL/6 mice (3 per group) were immunized on day 0 with either 1×10⁶ pfu of vaccinia virus (“VV”) encoding OVA or with 5×10⁶ cfu of recombinant Listeria monocytogenes (“Lm”) deleted of the actA and inlB virulence determinants and encoding OVA. On day 21 the mice received a boost dose of either the VV or Lmat doses that in each case were equivalent to the priming immunization doses. The mice were sacrificed and splenocytes harvested on day 27. To assess the magnitude of vaccine-induced OVA-specific CD8+ T cells after a single immunization, control mice received only a single immunization with VV or Lm on day 20 and were sacrificed and splenocytes harvested on day 27. OVA specific CD8+ T cell responses were determined using OVA₂₅₇₋₂₆₄ peptide in ISC assays.

The OVA specific CD8+ T cell immune responses resulting from the prime-boost regimens are shown in FIG. 1A. As seen in the figure, a prime-boost regimen utilizing a VV prime/Lm boost yields almost a doubling in the percentage of OVA specific CD8⁺ cells when compared to an LM prime/VV boost. In addition the percentage of OVA specific CD8⁺ cells in the VVprime/Lm boost is about 3 fold higher than a homologous Lm prime/Lm boost, and about 9 fold higher than a homologous VV prime/VV boost. This data provides evidence of directionality in the heterologous prime-boost regimen, with superior results obtained from a system that uses an Lm boost.

Example 2

Balb/c mice with 3 mice per group were injected on day 0 with either 3×10⁷ pfu adenovirus (“AV” or “adeno-hMeso”) encoding human Mesothelin or with 5×10⁶ cfu LmΔactA/ΔinlB encoding human Mesothelin (“Lm-hMeso38”). On day 21 the mice received a boost dose of either the AV or Lm-hMeso38 at doses that in each case were equivalent to the priming immunization doses. The mice were sacrificed and splenocytes harvested on day 27. To assess the magnitude of vaccine-induced OVA-specific CD8+ T cells after a single immunization, control mice received only the AV and Lm-hMeso38 on day 20 and were sacrificed and splenocytes harvested on day 27.

The vaccine-induced human Mesothelin specific CD4+ and CD8+ cellular immune responses resulting from the prime-boost regimens are shown in FIG. 1B. The AV prime/Lm-hMeso38 boost yielded about a 10-fold higher magnitude of the percentage of splenic CD8⁺ cells specific for human Mesothelin (“hMeso”) when compared to the cohort of mice given an Lm prime/AV boost regimen, demonstrating both that the directionality in the heterologous boosting regimen affects the magnitude of the vaccine-induced cellular immunity specific for the encoded antigen, and that superior results are obtained from a system that uses an Lm boost.

Example 3

Balb/c mice (3 per group) received priming doses and boosting doses to provoke an immune response against human Mesothelin. The priming and boosting regimen was as indicated in FIG. 2 using the indicated vectors that all encoded human Mesothelin. As seen from the results of the ICS assays, a regimen of AV prime/Lm boost yielded a 3 to 4 fold higher percentage of human Mesothelin (“hMeso”) specific CD8⁺ cells than did an Lm prime/AV boost. In addition, the results in FIG. 2 also show that the AV prime/Lm boost was significantly higher than any of the other heterologous prime-boost regimens tested.

Example 4

The effect of different AV priming doses against a constant Lm boost in the heterologous AV prime/Lm boost on the magnitude of vaccine-induced splenic hMeso-specific CD4+ and CD8+ T cell immunity was tested. All the vectors encoded human Mesothelin. HBSS was used as a control. The prime and boost regimen utilized was as indicated in FIG. 3A, with each experimental cohort consisting of three C57BL/6 mice. In these studies, the splenocytes obtained from the immunized mice were stimulated for 5 hours with a Mesothelin peptide pool library consisting of peptides 15 amino acids long offset by 4 amino acids (“15×11 library”) corresponding to the full-length Mesothlin protein prior to ICS. The human Mesothelin specific cellular immune responses resulting from the titration of AV in the prime-boost regimens are shown in FIG. 3A. As seen in the figure, significant enhancement of specific T cell responses was obtained at the higher amounts of AV, and the CD8⁺ Tcell response was greater than that of CD4⁺.

Example 5

An immunization protocol similar to that used in Example 4 was used in Balb/c mice (See FIG. 3B), except that the time between the prime and the boost was 38 days, and control mice were immunized with Lm hMeso 38, but not primed with AV. As seen in FIG. 3B, the significant enhancement of specific T cell responses were over a greater range of AV amounts, and again, the CD8⁺ Tcell response was greater than that of CD4⁺. In Balb/c mice, at AV priming doses as low as 1×10⁴ pfu substantial levels of hMeso-specific CD8+ T cells were induced after boosting with 5×10⁶ cfu of Lm hMeso 38.

Example 6

The effectiveness of an AV prime/Lm boost in the presence of pre-existing immunity to AV was tested. In the first study, the responsiveness to AV immunization in the presence of pre-existing adenovirus-specific immunity was tested using the protocol shown in FIG. 4A. Pre-existing adenovirus specific immunity was elicited by immunizing mice with various levels of “empty Ad vector” as shown in the FIG. 28 days prior to immunization with AV encoding human Mesothelin. The study used 5 Balb/C mice per group. The mice were injected on day 0 with either empty Ad particles and subsequently, on day 28 with AV encoding human Mesothelin (“Ad-hMeso”). Splenocytes were harvested on day 35 and used in ICS assays. The Ad-empty and Ad-hMeso vectors were both serotype 5. Mesothelin specific immune response was measured by stimulating splenocytes with human Mesothelin peptide pool; AV specific responses were measured using the class I Hex3 epitope. The results shown in FIG. 4A indicate that in the presence of pre-existing Ad5-specific cellular immunity only a low hMeso specific response could be induced when mice were immunized with the same Ad5 vector encoding human Mesothelin.

The second study tested the hMeso specific cellular immune response after an AVprime/Lm boost regimen in mice that had pre-existing AV immunity (shown as amount of AV pfu administrated on the X-axis of FIG. 4B). The protocol used (shown in FIG. 4B) was similar to that to test the prime, except that 20 days after the prime with AV, the mice were given a boost with Lm-hMeso38, and instead of measuring AV specific T cell immune response, AV-specific neutralizing antibodies were determined by plaque reduction assay. The results in FIG. 4B when compared to FIG. 4A show that despite the existence of pre-existing neutralizing immunity to AV serotype5, that combining a boost of Lm hMeso38 following an AV prime resulted in the induction of a significant increase in hMeso specific cellular immunity.

Example 7

A heterologous prime-boost using a tumor cell prime/Lm boost was performed as follows. The regimen used GVAX® (CELL GENESYS, INC.) prime/Lm-AH1-A5 boost. “GVAX” refers to an inactivated tumor cell containing a nucleic acid that encodes granuclocyte macrophage-colony stimulation factor (GM-CSF). The administered Listeria monocytogenes was Lm-ΔactA-OVA-AH1-A5. Lm-ΔactA-OVA-AH1-A5 is a recombinant Listeria monocytogenes, attenuated by a deletion in the actA gene, and containing a single copy of an antigen expression cassette in the listerial genome, integrated at the tRNA^(Arg) locus, where the expression cassette contains the AH1-A5 inserted in-frame within ovalbumin (Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101:13832-13837). AH1-A5, is the altered T cell ligand of the AH1 L^(d) immunodominant epitope of the gp70 endogenous rejection antigen expressed by the CT26 adenocarcinoma cell line, and is conventionally used in studies of immune response to the tumor antigen, gp70 (Slansky, et al. (2000) Immunity 13:529-538; Jain, et al. (2003) Annals Surgical Oncol. 10:810-820).

For the heterologous prime/boost, 3 mice in each group received GVAX® (CELL GENESYS, INC.) (1×10⁶ cells) (s.c.) as a prime and Lm-OVA-AH1-A5 (5×10⁶ cfu) (i.v.) as a boost. For homologous prime/boost vaccines with GVAX, mice received a GVAX® (CELL GENESYS, INC.) prime (1×10⁶ cells) (s.c.), for both the prime and boost. For homologous prime/boost vaccines with Lm only, mice were given Lm-OVA-AH1-A5 (5×10⁶ cfu) (i.v.), for both the prime and boost. Separate homologous prime/boost trials were conducted, where Lm was only administered s.c., i.m., or i.v. In all cases, the prime was administered at t=0 days, boost at t=21 days, with harvest of splenocytes at t=29 days. Mice were also treated with single-administration mono-vaccines (GVAX® (CELL GENESYS, INC.) only; Lm-OVA-AH1-A5 only), as indicated in the figure.

The results demonstrate that GVAX® (CELL GENESYS, INC.) prime followed by an Lm-ΔactA-OVA-AH1-A5 boost elicited greater AH1-specific immune responses than any homologous prime/boost immunization regimen by any of the routes of administration.

Example 8

A comparison of AV prime/Lm boost with dendritic cell (“DC”) prime/Lm boost was performed as follows. Balb/c mice, 5 mice per group, were used. Dendritic cells were administered in the quantity of 2×10⁶ dendritic cells (i.v.). Adenovirus-derived vector containing a nucleic acid encoding human Mesothelin was administered at a does of 1×10⁸ cfu (i.m.). Lm-hMeso38 was administered in the quantity of 5×10⁶ cfu (i.v.). Peptide-pulsed DCs were administered at a dose of 2×10⁶ DC (i.v.). DC and Lm prime and boost immunizations were separated by 8 days, and AV and Lm prime and boost immunizations were separated by 14 days. In all mice, the splenic Mesothelin 131-139 specific CD8+ T cells were measured by days following the boost immunization with Lm hMeso 38. Controls were as indicated in FIG. 6A and FIG. 6B.

Peptide-pulsed DCs were prepared as follows. The DCs were pulsed with hMeso₁₃₁₋₁₃₉ (SGPQACTRF). DC were prepared from whole bone marrow of Balb/c mice using high GM-CSF concentrations (20 ng/mL murine GM-CSF) (R&D Systems, Minneapolis, Minn.). On day 8 following initial plating and GM-CSF enrichment, non-adherent cells were harvested and verified phenotypically to be myeloid dendritic cells (CD11c^(hi)). The DCs were treated with lipopolysaccharide (LPS) (24 h) and pulsed with 1.0 micromolar peptide (hMeso₁₃₁₋₁₃₉) (0.001 mM) for 1 hour. Peptide-loaded DCs were washed two times, before injecting 1×106 DCs into recipient Balb/c mice.

The results of the ICS assays presented in FIG. 6A demonstrate that heterologous prime/boost with DC-hMeso131-139 prime/Lm-hMeso38 boost, and heterologous Adeno-hMeso prime/Lm-hMeso38 boost, each induced Mesothelin-specific cellular immune responses, where the level of immune response was about the same for both prime/boost regimens.

The results of Elispot assays, shown in FIG. 6B, demonstrate that heterologous prime/boost with DC-hMeso131-139 prime/Lm-hMeso38 boost, and heterologous Adeno-hMeso prime/Lm-hMeso38 boost, each induced Mesothelin-specific cellular immune responses, where the level of immune response was about the same for both prime/boost regimens.

Example 9

A comparison of Lm, AV, and VV as a boost after a DC prime was performed as follows. On day 0, Balb/cmice (five per group) were vaccinated intravenously with 1×10⁶ bone marrow-derived dendritic cells (prime). The DC were prepared as described in Example 8. At 21 days, the mice were provided with a boost, where the boost was one of the following: Lm-hMeso38 (5×10⁶ cfu, i.v.), Ad-hMeso (1×10⁷ pfu, i.m.) or vaccinia virus-hMeso (“VV-hMeso”)(1×10⁶ pfu, i.p.). A separate group of mice received HBSS instead of the boost vaccine. Meso₁₃₁₋₁₃₉-specific CD8⁺ T cell responses were determined by intracellular cytokine staining (ICS) five days following the boost vaccination.

The results shown in FIG. 6C indicate that although the AV-hMeso did give a significant boost to the hMeso cell specific response, approximately a five-fold higher result was achieved using Lm as the boosting agent.

Example 10

A comparison of AV and naked DNA as the prime in a heterologous prime-boost regimen using Lm as the boost was performed. In addition, mouse Mesothelin was substituted for human Mesothelin to determine if it was possible to break tolerance with the heterologous prime-boost regimen. In the study, Balb/c mice were used. Heterologous DNA prime/Lm boost and heterologous Ad prime/Lm boost vaccines were administered, with assessment of Mesothelin-specific immune response using the entire Mesothelin peptide library. The DNA prime/Lm boost was DNA-mMeso (0.1 mg) (i.m.) prime/Lm-mMeso38 (5×10⁶ cfu) (i.v.) boost (FIG. 7A). The Ad prime/Lm boost was Ad-mMeso (1×10⁸ pfu) (i.m.) prime/Lm-mMeso38 (5×10⁶ cfu) (i.v.) boost (FIG. 7B). The naked DNA vector, DNA-mMeso, was prepared as follows. A nucleic acid encoding full length mouse mesothlin was inserted into pcDNA3, a eukaryotic expression vector (Invitrogen Corp., Carlsbad, Calif.).

The prime was administered at day 0, boost at 13 days, with harvest of splenocytes at 18 days. Harvested splenocytes were treated with peptides from the mouse Mesothelin (“mMesothelin”) peptide library, where a unique peptide was added to each well containing splenocytes, and where immune response was assessed with Elispot assays. Sufficient wells of splenocytes from immunized mice were utilized such that unique peptides (15 amino acids long, offset by 4 amino acids) covering the full length of the mouse Mesothelin protein could be used, enabling the T cell reactive mouse Mesothein peptides to be identified.

The results in FIG. 7A indicate that when the immune response was separately assessed for each peptide in the mMesothelin peptide library, the DNA-mMeso prime/Lm-mMeso38 boost elicited relatively little immune response. By contrast, the results in FIG. 7B indicate that the Ad-mMeso prime/Lm-mMeso38 boost induced relatively high immune response to mouse Mesothelin peptides nos. 278, 279, and 280, from the mouse Mesothelin peptide library. Relatively little immune response was found where other peptides from the mouse Mesothelin peptide library had been used. FIG. 7C is a photograph of the wells, where splenocytes had been exposed to Mesothelin peptide nos. 278, 279, or 280, and where the photograph illustrates the spots from the elispot assays. Thus, the results shown in FIG. 7B and FIG. 7C demonstrate that the AVprime/Lm boost regimen resulted in a specific cellular immune response to mMesothelin. Moreover, the results also indicate the breaking of tolerance against the mouse Mesothelin endogenous antigen in mice given a prime and boost immunization regimen consisting of AV and Lm vectors, with both vectors encoding mouse Mesothelin.

Example 11

The efficacy of KBMA Lm as a priming agent and/or a boosting agent was evaluated. C57BL/6 mice (3 per group) were administered with KBMA-Lm as a prime at day 0. A boost of one of the following was given at day 14: “Live” Lm, KBMA-Lm, or VV. Controls of KBMA-Lm and Live Lm were also administered at day 14. The mice were sacrificed on day 19, and splenocytes were harvested.

The Listeria constructs had a hly promoter operably linked to BaPA secretory sequence and ovalbumin, with genomic integration at the tRNAArg locus.

The number of KBMA Listeria monocytogenes ΔactAΔuvrAB-OVA (KBMA Lm) injected was 3×10⁸ bacterial particles. The KBMA Lm had been previously treated with psoralen and UV light. The amount of administered “Live” Listeria monocytogenes (Lm-ΔactAΔuvrAB-OVA) was 1×10⁶ cfu. The number of administered vaccinia virus-derived vector (VV-OVA) was 1×10⁶ pfu.

Immune response was determined by peptide-pulsing the splenocytes with a standard octapeptide peptide derived from ovalbumin (OVA₂₅₇₋₂₆₄), and measuring ovalbumin-specific immune response by way of intracellular staining (ICS) assays for IFN-γ.

As seen from the results, shown in FIG. 8, KBMA-Lm was active as a priming agent. The KBMA-Lm prime/live Lm boost elicited the highest level of immune response, where lesser levels of immune response occurred with the heterologous KBMA-Lm prime/VV boost vaccine.

Example 12

Fifteen Balb/c mice per group were vaccinated intramuscularly with 1e8 pfu of empty adenovirus (i.e., adenovirus not expressing AH1, AH1/A5, or mMesothelin) or adenovirus expressing AH1/A5 or murine Mesothelin (mMesothelin). 21 days later, the mice were boosted intravenously with 5e6 cfu of Lm not encoding a heterologous antigen or Lm expressing AH1/A5 or mMesothelin. AH1/A5 is described (see, e.g., Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101:13832-13837). Spleens from five mice per group were harvested after five days to assess AH1/A5 (FIG. 9A) and mMesothelin (FIG. 9B) immune responses. One week after the boost vaccination, the remaining mice were challenged with 4e5 CT26 cells intravenously. Mice were monitored for survival (FIG. 9C). In FIGS. 9A-9C, “Ad” denotes empty adenovirus, “Lm” denotes L. monocytogenes not encoding a heterologous antigen, “Ad-AH1/A5” denotes adenovirus expressing AH1/A5, “Ad-mMeso” denotes adenovirus expressing murine Mesothelin, “Lm-AH1/A5” denotes L. monocytogenes encoding AH1/A5, and “Lm-mMeso” denotes L. monocytogenes encoding murine Mesothelin.

As shown in FIG. 9A, prime/boost with Ad-AH1/A5 prime and Lm-AH1/A5 boost resulted in substantial antigen-specific CD8+ T cell immune response, according to ICS assays. AH1 peptide and AH1/A5 peptide are derived from gp70, an endogenous protein of CT26 tumor cells. Splenocyte incubation mixtures were supplemented with the AH1 peptide or AH1/A5 peptide, as indicated. Gp70 is an endogenous protein of CT26. CD8+ T cell immune response was greater where splenocytes were incubated with added AH1/A5 peptide, and lesser with added AH1 peptide.

Specificity of the immune response was demonstrated as follows. Prime/boost with empty Adenovirus and Lm not encoding a heterologous Ag did not result in detectable CD8+ T cell immune response. Also, prime/boost with vectors expressing murine Mesothelin did not result in detectable AH- or AH1/A5-specific CD8+ T cell immune responses, again demonstrating specificity of the immune response (FIG. 9A).

The data shown in FIG. 9B demonstrate prime/boost stimulation of an immune response against another heterologous antigen, namely Mesothelin, and that the immune response was specific for this antigen. Specificity was demonstrated by the failure of empty Ad prime/Lm (not encoding heterologous Ag) boost, or Ad AH1/A5 prime/Lm AH1/A5 boost, to stimulate mMesothelin specific CD8+ T cell immune responses.

The data shown in FIG. 9C demonstrate that the Ad-AH1/A5 prime/Lm-AH1/A5 boost increases survival to challenge with CT26 tumor cells. The figure also demonstrates that Ad-mMeso prime/Lm-mMeso increases survival to the CT26 tumor cell challenge. Prime boost treatment with empty vectors (empty Ad; Lm not encoding heterologous Ag) did not alter survival, as compared to treatment with HBSS (FIG. 9C).

Example 13

The investigational agent, CRS-207, consists of a live-attenuated strain of the intracellular bacterium Listeria monocytogenes (LM) that encodes the tumor antigen Mesothelin (Lm ΔactA/ΔinlB/hMeso). Administration of CRS-207 aims to induce anti-tumor response by induction of cell-mediated immunity (CMI) directed against mesothelin expressed on the cell surface of cancers such as malignant mesothelioma, non-small cell lung carcinoma (NSCLC), and carcinomas for the pancreas and ovary. CRS-207 was derived by deleting the entire coding sequences of two virulence-determinant genes from the genome of wild-type Lm. The products of the two virulence-determinant genes, inlB and actA, facilitate Lm invasion of non-phagocytic cells and cell-to-cell spread, respectively. The combined deletion of these two coding sequences causes attenuation by more than 1,000-fold, as assessed by virulence in mice. However, uptake of CRS-207 by macrophages and other phagocytic cells in the liver and spleen is retained and results in a local inflammatory response and activation and recruitment to the liver of immune effector cells such as natural killer (NK) cells and T cells. After uptake of CRS-207 by phagocytic cells (including DC and macrophages), Mesothelin is expressed and released into the cytosolic compartment and subsequently processed through the endogenous MHC class I presentation pathway, resulting in activation of anti-Mesothelin cell-mediated immunity (CMI). Other mechanisms to activate Mesothelin-specific CMI may include uptake and cross-presentation of antigens by APC from macrophages and/or other cell types after infection by CRS-207 and apoptosis.

CRS-207 was formulated at two concentrations: High Dose and Low Dose. Two concentrations were selected to facilitate preparation of the range of doses anticipated in the clinical study. See Tables 1a and 1b for the formulations of the CRS-207 investigational agents.

TABLE 1a CRS-207 High Dose Formulation Component Concentration Attenuated Listeria monocytogenes 5 × 10¹⁰ cfu/mL (ΔactA/ΔinlB strain hMeso38) Dulbecco's Phosphate Buffered Saline, containing: Sodium chloride, USP 8 g/L Potassium chloride, USP 0.2 g/L Sodium phosphate dibasic, USP 1.15 g/L Potassium phosphate monobasic, 0.2 g/L NF/FCC/EP/BP Glycerol, USP 9% (v/v)

TABLE 1b CRS-207 Low Dose Formulation Component Concentration Attenuated Listeria monocytogenes 1 × 10⁸ cfu/mL (ΔactA/ΔinlB strain hMeso38) Dulbecco's Phosphate Buffered Saline, containing: Sodium chloride, USP 8 g/L Potassium chloride, USP 0.2 g/L Sodium phosphate dibasic, USP 1.15 g/L Potassium phosphate monobasic, 0.2 g/L NF/FCC/EP/BP Glycerol, USP 9% (v/v)

CRS-207 stored frozen at −75° C. or colder. It consists of attenuated Lm suspended in 1.5 mL of Dulbecco's phosphate buffered saline (DPBS) and 9% v/v glycerol. Each high dose vial has a concentration of 5×10¹° cfu/mL and each low dose vial has a concentration of 1×108 cfu/mL.

CRS-207 was delivered to human subjects (≧18 years old) with advanced carcinoma of the ovary or pancreas, non-small cell lung cancer or malignant mesothelioma who had failed standard therapy or who were not candidates for standard therapy. Subjects met all of the following inclusion criteria for enrollment in the study:

-   -   Histologically or cytologically documented malignant         mesothelioma, adenocarcinoma of the pancreas, or         Mesothelin-expressing non-small-cell lung carcinoma (NSCLC) or         carcinoma of the ovaries who have failed or who are not         candidates for standard treatment     -   ECOG performance score 0-1 or Karnofsky Performance Status (KPS)         80% to 100%     -   Anticipated life expectancy greater than the duration of the         study     -   Female subjects of childbearing potential and all male subjects         must consent to use a medically acceptable method of highly         effective contraception throughout the study period and for 28         days after CRS 207 administration (oral hormonal contraceptive,         condom plus spermicide, or hormone implants) by all subjects. A         barrier method of contraception must be included, regardless of         other methods.     -   Subject provides informed consent and is willing and able to         comply with all study procedures.     -   Adequate organ function defined as follows:

Hematologic

-   -   Platelets≧100×10⁹/L     -   Hemoglobin≧9.0 g/dL     -   Total WBC count≧3.5×10⁹/L     -   ANC≧1.5×109/L     -   Total lymphocyte count >0.8×10⁹/L     -   PT/INR and PTT ≦1.3× clinical laboratory ULN

Hepatic

-   -   Bilirubin ≦1.5× clinical laboratory ULN     -   AST and ALT ≦2.5× clinical laboratory ULN (≦3.5× clinical         laboratory ULN is acceptable for patients with pancreatic         adenocarcinoma)     -   GGT ≦5× clinical laboratory ULN     -   Alkaline phosphatase ≦2.5× clinical laboratory ULN

Renal

-   -   Serum creatinine <1.5× clinical laboratory ULN

In addition, the subject must not have any of the following exclusion criteria:

-   -   Known metastases to central nervous system.     -   History of listeriosis or vaccination with a listeria-based         vaccine.     -   Known allergy to penicillin.     -   Clinically significant heart disease (such as uncontrolled         angina, myocardial infarction within 3 months, congestive heart         failure of New York Heart Association III or IV).     -   Individuals with valvular heart disease who require antibiotic         prophylaxis for prevention of endocarditis, consistent with AHA         guidelines (Dajani et al. Circulation, 1999).     -   O2 saturation <92% on room air, as measured by pulse oximeter     -   Study subjects who have or are suspected to have compromised         pulmonary function, based upon history and physical exam, are         not eligible for study entry unless VC, DLCO, and FEV1 are         documented >60% of predicted value.     -   Active, clinically significant, autoimmune disease or history of         autoimmune disease requiring systemic treatment.     -   Clinical laboratory results corresponding to Grade 3 or 4 CTCAE         clinical metabolic/laboratory abnormalities.     -   Hepatic cirrhosis or clinically relevant ascites or individuals         who have substantial risk of obstructive jaundice (e.g., by         tumor mass compression of the hepatic hilum).     -   Artificial (prosthetic) joint or other artificial implant or         devices that cannot easily be removed. Dental implants and         breast implants are allowed if there is no history of infection         of the implants and no clinically significant adverse events         associated with the implants.     -   Known coagulation disorder or receiving anticoagulant medication     -   Patients requiring regular transfusions more frequently than         twice per month.     -   Blood transfusion within fourteen days of study entry, unless         leuko-reduced and irradiated.     -   Any immunodeficiency disease or immune compromised state (e.g.,         use of immunosuppressive agents; chemotherapy or radiation         therapy within 28 days prior to CRS 207 administration or         planned within 28 days after CRS 207 administration).     -   Use of systemically active steroids for more than 2 days, within         28 days prior to CRS 207 administration or planned within the         study period after CRS 207 administration. Use of any systemic         steroids within 14 days of CRS 207 administration.     -   Female subjects who are pregnant or lactating. Female subjects         of childbearing potential must have a negative β-hCG test (serum         or urine).     -   History of alcohol dependence or use of illicit drugs that could         potentially interfere with adherence to study procedures or         requirements (e.g., opioids, cocaine, amphetamines,         hallucinogens, etc.)

Subjects were treated with CRS 207 administered intravenously over 2 hours. The dose was increased for each successive dose group as shown in the table, below:

Dose Cohort Dose (cfu) 1 1 × 10⁸  2 1 × 10⁹  3 1 × 10¹⁰ 4 3 × 10¹⁰ During the dose escalation portion of the study, CRS-207 will be administered with 21 day intervals between doses.

Subjects were grouped into those subjects having a survival ≧15 months following first dosage with CRS 207 and those having a survival <15 months following first dosage with CRS 207. The following table shows the demographics in these two groups:

survival ≧15 survival <15 months months Age at Consent (mean ± 59.8 ± 3.2   62 ± 8.4 SD) Race 100% White 100% White Ethnicity 100% Not Hispanic 100% Not Hispanic or Latino or Latino Gender 25% Male 55% Male 75% Female 45% Female % of Subjects who Met All 50% 82% Protocol Criteria Cancer Type 50% Pancreatic 36% Pancreatic 33% NSCLC 36% Mesothelioma 17% Mesothelioma 18% Ovarian 9% NSCLC % of Subjects with 0 ECOG 83% 36% status prior to study # of Prior Therapies 4.0 ± 0.7 3.5 ± 2.5 (mean ± SD) # of Subjects, By Cohort: 1 × 10⁸ CFU 3 3 1 × 10⁹ CFU 2 4 1 × 10¹⁰ CFU 0 1 3 × 10⁸ CFU² 1 3 # of Doses Received 3.5 ± 0.8 2.6 ± 0.9 (mean ± SD) % of Subjects Receiving All 83% 27% 4 Doses % of Subjects w/vaccine- 100% (5/5)  0% (3/3) induced LLO-specific immune response % of Subjects w/primary 100% (4/4) 100% (3/3) tumor tissue mesothelin positive by IHC Survival from 1^(st) dose of 19.4 ± 3.0  4.5 ± 1.6 CRS-207 as of Apr. 15, 2010, months (mean ± SD)

Survival was improved in those subjects for whom CRS 207 was an adjuvant therapy:

survival ≧15 survival <15 months months % with Prior Immunotherapy 67% 9% % with Prior Radiation 83% 9% % with Prior Surgery 83% 73% 

In those subjects who had previously received GVAX® (CELL GENESYS, INC.) in a phase II trial, survival was improved by subsequent delivery of CRS 207:

Subjects who Subjects Subsequently who did not All Received Receive Subjects CRS-207 CRS-207 # of Subjects 13 2 11 % of Subjects who are 31% (4/13) 50% (1/2) 27% (3/11) HLA-A2 % of Subjects who Received 62% (8/13)  0% (0/2) 73% (8/11) Erbitux (i.e., did not have hypersensitive rxn) Survival from Diagnosis, 26.3 ± 12.1 43.0 ± 2.0 23.3 ± 9.9 months (mean ± SD) Survival from Consent, 12.2 ± 6.4  28.0 ± 8.0  9.3 ± 3.4 months (mean ± SD)

One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

While the invention has been described and exemplified in sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.

It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

Other embodiments are set forth within the following claims. 

What is claimed:
 1. A method for adjuvant treatment of a mammal suffering from cancer, comprising: administering to the mammal an effective amount of a composition comprising an attenuated, metabolically active Listeria that encodes an expressible, immunologically active portion of a cancer antigen wherein the mammal previously had been administered an effective amount of radiation therapy as a primary therapy administered to the mammal to kill cancer cells expressing the cancer antigen, wherein prior to the administering step, the mammal previously had been administered cells from a human cell line expressing all or a portion of the cancer antigen, wherein said cells have been recombinantly modified to produce and secrete granulocyte macrophage colony stimulating factor, and wherein said cells have been modified to prevent the cells from dividing.
 2. A method according to claim 1, wherein the administration step comprises administering the attenuated, metabolically active Listeria that encodes an expressible, immunologically active portion of a cancer antigen in multiple doses.
 3. The method according to claim 1 wherein the attenuated, metabolically active Listeria has a mutation that inactivates ActA.
 4. The method according to claim 3 wherein the attenuated, metabolically active Listeria has a mutation that inactivates InlB.
 5. The method according to claim 4 wherein the attenuated, metabolically active Listeria is ΔactAΔinlB.
 6. The method according to claim 1 wherein the Listeria is killed but metabolically active (“KBMA”).
 7. A method according to claim 1 wherein the cancer antigen is all or a portion of mesothelin.
 8. A method according to claim 7 wherein said cancer is selected from the group consisting of pancreatic cancer, non-small cell lung cancer, ovarian cancer, and mesothelioma. 